Live bioload detection using microparticles

ABSTRACT

The present invention provides methods to concentrate cells onto microparticles, to concentrate the microparticles, and to detect the cells. The present invention also includes unitary sample preparation and detection devices to be used in accordance with the methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/141,685, filed Dec. 31, 2008 and U.S. ProvisionalPatent Application No. 61/291,301, filed Dec. 30, 2009, which areincorporated herein by reference.

BACKGROUND

Various tests are available that can be used to assess the presence ofbiological analytes in a sample (e.g. surface, water, air, etc). Suchtests include those based on the detection of ATP using the fireflyluciferase reaction, tests based on the detection of protein usingcolorimetry, tests based on the detection of microorganisms usingmicrobiological culture techniques, and tests based on detection ofmicroorganisms using immunochemical techniques. Surfaces can be sampledusing either a swab device or by direct contact with a culture devicesuch as an agar plate. The sample can be analyzed for the presence oflive cells and, in particular, live microorganisms.

Results from these tests are often used to make decisions about thecleanliness of a surface. For example, the test may be used to decidewhether food-processing equipment has been cleaned well enough to usefor production. Although the above tests are useful in the detection ofa contaminated surface, they can require numerous steps to perform thetest, they may not be able to distinguish quickly and/or easily thepresence of live cells from dead cells and, in some cases, they canrequire long periods of time (e.g., hours or days) before the resultscan be determined.

The tests may be used to indicate the presence of live microorganisms.For such tests, a cell extractant is often used to release a biologicalanalyte (e.g., ATP) associated with living cells. The presence ofextracellular material (e.g., non-cellular ATP released into theenvironment from dead or stressed animal cells, plant cells, and/ormicroorganisms) can create a high “background” level of ATP that cancomplicate the detection of live cells.

In spite of the availability of a number of methods and devices todetect live cells, there remains a need for a simple, reliable test fordetecting live cells and, in particular, live microbial cells.

SUMMARY OF THE INVENTION

In general, the present disclosure relates to articles and methods fordetecting live cells in a sample. The articles and methods make possiblethe rapid detection (e.g., through fluorescence, chemiluminescence, or acolor reaction) of the presence of cells such as bacteria on a surface.In some embodiments, the inventive articles are “sample-ready”, i.e.,the articles contain all of the necessary features to detect livingcells in a sample. In some aspects, the inventive articles and methodsprovide a means to distinguish a biological analyte, such as ATP or anenzyme, that is associated with eukaryotic cells (e.g., plant or animalcells) from a similar or identical biological analyte associated withprokaryotic cells (e.g., bacterial cells). Furthermore, the inventivearticles and methods provide a means to distinguish a biological analytethat is free in the environment (i.e., an acellular biological analyte)from a similar or identical biological analyte associated with a livingcell.

Methods of the present disclosure allow an operator to concentrate cellsfrom a liquid sample and to detect an analyte associated with the cells.In certain embodiments, detection of the analyte may be an indicator oflive cells including, in particular, live microbial cells in the sample.In some embodiments, the methods provide for the operator to measure theamount of a biological analyte in the sample. In some embodiments, themethods provide for the operator to, after a predetermined period oftime during which an effective amount of a cell extractant is releasedfrom a composition into the liquid mixture, measure the amount of abiological analyte to determine differentially the amount of biologicalanalyte from acellular material and from live cells in the sample. Insome embodiments, the methods provide for the operator, within a firstpredetermined period of time, to perform a first measurement of theamount of a biological analyte and, within a second predetermined periodof time during which an effective amount of cell extractant is releasedfrom the composition, perform a second measurement of the amount ofbiological analyte to detect the presence of live cells in the sample.In some embodiments, the methods can allow the operator to distinguishwhether biological analyte in the sample was released from live plant oranimal cells or whether it was released from live microbial cells (e.g.,bacteria). The present invention is capable of use by operators underthe relatively harsh field environment of institutional food preparationservices, health care environments and the like.

In one aspect, the present disclosure provides a method of detectingcells in a sample. The method comprises providing a cell concentrationagent, a hydrogel comprising a cell extractant and a liquid samplesuspected of containing cells. The method further comprises contactingthe liquid sample and the cell concentration agent for a period of time,isolating the cell concentration agent from at least a portion of theliquid sample, forming a liquid mixture comprising the isolated cellconcentration agent and the hydrogel wherein the cell extractant isreleased into the mixture, and detecting a biological analyte.Optionally, the analyte can be detected at two or more discrete timepoints. In some embodiments, detecting a biological analyte comprisesdetecting a live cell. In some embodiments, detecting a biologicalanalyte comprises using a detection system. In some embodiments,detecting a biological analyte comprises quantifying the analyte. Insome embodiments, detecting a biological analyte comprises detecting ATPfrom a cell. In some embodiments, detecting a biological analytecomprises detecting the cell by genetic or immunological methods. Insome embodiments, the method further comprises the steps of providing asomatic cell extractant and contacting the somatic cell extractant withcells from the sample.

In another aspect, the present disclosure provides a method of detectingcells in a sample. The method comprises providing a sample suspected ofcontaining cells; a cell concentration agent; a hydrogel comprising acell extractant; a detection article comprising a housing with two ormore receptacles and an opening configured to receive the sample; meansfor isolating and transferring the cell concentration agent from a upperreceptacle to a lower receptacle in the housing. The method furthercomprises contacting in a liquid medium the sample and the cellconcentration agent in the upper receptacle of the housing. The methodfurther comprises transferring the cell concentration agent to the lowerreceptacle in the housing. The method further comprises forming a liquidmixture comprising the isolated cell concentration agent and thehydrogel, wherein the cell extractant is released into the mixture. Themethod further comprises detecting a biological analyte. Optionally, thebiological analyte can be detected at two or more discrete time points.In some embodiments, detecting a biological analyte comprises detectinga live cell. In some embodiments, detecting a biological analytecomprises using a detection system. In some embodiments, detecting abiological analyte comprises quantifying the analyte. In someembodiments, detecting a biological analyte comprises detecting ATP froma cell. In some embodiments, detecting a biological analyte comprisesdetecting the cell by genetic or immunological methods. In someembodiments, the method further comprises the steps of providing asomatic cell extractant and contacting the somatic cell extractant withcells from the sample.

In another aspect, the present disclosure provides a method of detectingcells in a sample. The method comprises providing a sample suspected ofcontaining cells; a detection article comprising a housing with anopening configured to receive the sample, a upper receptacle containinga cell concentration agent, and a lower receptacle containing a hydrogelcomprising a cell extractant; means for isolating the cell concentrationagent from at least a portion of the liquid sample; and means fortransferring the cell concentration agent from the upper receptacle tothe lower receptacle in the housing. The method further comprisescontacting in a liquid medium the sample and the cell concentrationagent in the upper receptacle of the housing. The method furthercomprises isolating and transferring the cell concentration agent to thelower receptacle of the housing. The method further comprises forming aliquid mixture comprising the isolated cell concentration agent and thehydrogel, wherein the cell extractant is released into the mixture. Themethod further comprises detecting a biological analyte. Optionally, thebiological analyte can be detected at two or more discrete time points.In some embodiments, detecting a biological analyte comprises detectinga live cell. In some embodiments, detecting a biological analytecomprises using a detection system. In some embodiments, detecting abiological analyte comprises quantifying the analyte. In someembodiments, detecting a biological analyte comprises detecting ATP froma cell. In some embodiments, detecting a biological analyte comprisesdetecting the cell by genetic or immunological methods. In someembodiments, the method further comprises the steps of providing asomatic cell extractant and contacting the somatic cell extractant withcells from the sample.

In another aspect, the present disclosure provides a unitary samplepreparation and detection device. The device comprises a housingcomprising at least two receptacles with a passageway therebetween. Aupper receptacle of the housing comprises an opening configured toreceive a sample and a cell concentration agent disposed therein. Alower receptacle of the housing comprises a detection reagent disposedtherein. The device further comprises means for isolating the upperreceptacle from the lower receptacle. The device further comprises meansfor transferring the cell concentration agent from the upper receptacleto the lower receptacle. In some embodiments, the means for isolatingthe first and lower receptacles is the means for transferring the cellconcentration agent from the upper receptacle to the lower receptacle.In some embodiments, the housing further comprises a frangible sealbetween the two isolated receptacles. In some embodiments, the upperreceptacle comprises a taper region. In some embodiments, the devicefurther comprises a hydrogel comprising a cell extractant. In someembodiments, the housing further comprises a third receptacle. In someembodiments, the device further comprises a sample acquisition device.In some embodiments, the detection reagent comprises a reagent fordetecting ATP. In some embodiments, the device further comprises ahydrogel comprising a detection reagent.

In another aspect, the present disclosure provides a unitary samplepreparation and detection device. The device comprises a housingcomprising at least two isolated receptacles with a passagewaytherebetween and a piston configured to fit the passageway. A upperreceptacle in the housing comprises an opening configured to receive asample and a cell concentration agent disposed therein. A lowerreceptacle of the housing comprises a detection reagent disposedtherein. In some embodiments, the housing further comprises a frangibleseal between the two isolated receptacles. In some embodiments, theupper receptacle comprises a tapered inner wall. In some embodiments,the device further comprises a hydrogel comprising a cell extractant. Insome embodiments, the housing further comprises a third isolatedreceptacle. In some embodiments, the device further comprises a sampleacquisition device. In some embodiments, the detection reagent comprisesa reagent for detecting ATP. In some embodiments, the device furthercomprises a slow-release composition comprising a detection reagent.

In another aspect, the present disclosure provides a unitary samplepreparation and detection device. The device comprises a housingcomprising at least two isolated receptacles with a passagewaytherebetween. An upper receptacle of the housing comprises an openingconfigured to receive a sample and a cell concentration agent disposedtherein. A lower receptacle comprises a detection reagent disposedtherein. The device further comprises a valve configured to control thepassage of material from the upper receptacle to the lower receptacle.In some embodiments, the upper receptacle comprises a tapered innerwall. In some embodiments, the device further comprises a hydrogelcomprising a cell extractant. In some embodiments, the housing furthercomprises a third isolated receptacle. In some embodiments, the devicefurther comprises a sample acquisition device. In some embodiments, thedetection reagent comprises a reagent for detecting ATP. In someembodiments, the device further comprises a slow-release compositioncomprising a detection reagent.

In another aspect, the present disclosure provides a kit comprising ahousing comprising at least two isolated receptacles with a passagewaytherebetween and means for transferring the cell concentration agentfrom an upper receptacle to a lower receptacle. The upper receptacle ofthe housing comprises an opening configured to receive a sample. Thelower receptacle comprises a detection reagent disposed therein. The kitfurther comprises a cell concentration agent. In some embodiments, thecell concentration agent is disposed in the upper receptacle of thehousing. In some embodiments, the kit further comprises hydrogelcomprising a microbial cell extractant. In some embodiments, the kitfurther comprises a somatic cell extractant. In some embodiments, thekit further comprises a sample acquisition device.

In another aspect, the present disclosure provides a kit comprising ahousing comprising at least two isolated receptacles with a passagewaytherebetween. An upper receptacle in the housing comprises an openingconfigured to receive a sample. A lower receptacle in the housingcomprises a detection reagent disposed therein. The kit furthercomprises a cell concentration agent and means for transferring the cellconcentration agent from the upper receptacle to the lower receptacle.In some embodiments, the cell concentration agent is disposed in theupper receptacle of the housing. In some embodiments, the kit furthercomprises hydrogel comprising a microbial cell extractant. In someembodiments, the kit further comprises a somatic cell extractant. Insome embodiments, the kit further comprises a sample acquisition device.

GLOSSARY

“Biological analytes”, as used herein, refers to molecules, orderivatives thereof, that occur in or are formed by an organism. Forexample, a biological analyte can include, but is not limited to, atleast one of an amino acid, a nucleic acid, a polypeptide, a protein, anucleotide, a polynucleotide, a lipid, a phospholipid, a saccharide, apolysaccharide, and combinations thereof. Specific examples ofbiological analytes can include, but are not limited to, a metabolite(e.g., a small molecule, such as ATP, or a polypeptide, such as proteinA), an allergen (e.g., peanut allergen(s), a hormone, a toxin (e.g.,Bacillus diarrheal toxin, aflatoxin, etc.), RNA (e.g., mRNA, total RNA,tRNA, etc.), DNA (e.g., plasmid DNA, plant DNA, etc.), a tagged protein,an antibody, an antigen, and combinations thereof.

“Liquid sample”, as used herein, refers to a sample material thatcomprises a liquid. The sample may, in its original form, comprise aliquid such as, for example, water, milk, juice, blood, wound exudate,and the like. Alternatively, the liquid sample can be a suspension ofsolids in a liquid suspending medium (e.g., water, an aqueous buffer).For example, a solid, semisolid, or gelatinous sample can be collectedwith a sample acquisition device and suspended in a liquid to form aliquid sample.

“Clarified liquid sample” refers to the bulk of a liquid sample thatremains after the liquid sample has been contacted with a cellconcentration agent and the cell concentration agent has beenpartitioned (e.g., by sedimentation, filtration, centrifugation, orprecipitation) from the bulk of the liquid.

“Sample acquisition device” is used herein in the broadest sense andrefers to an implement used to collect a liquid, semisolid, or solidsample material. Nonlimiting examples of sample acquisition devicesinclude swabs, wipes, sponges, scoops, spatulas, pipettes, pipette tips,and siphon hoses.

“Dead-end valve”, as used herein, refers to a type of valve that is usedto regulate the transfer of material (e.g., liquids, solids, or asuspension of solids in a liquid) between two or more receptacles in thehousing of a detection device. The dead-end valve is designed such thatthe cavity in the valve that is used to transfer the material can onlybe in fluid communication with one of the receptacles at a time.

As used herein, the term “hydrogel” refers to a polymeric material thatis hydrophilic and that is either swollen or capable of being swollenwith a polar solvent. The polymeric material typically swells but doesnot dissolve when contacted with the polar solvent. That is, thehydrogel is insoluble in the polar solvent. The swollen hydrogel can bedried to remove at least some of the polar solvent.

“Cell extractant”, as used herein, refers to any compound or combinationof compounds that alters cell membrane or cell wall permeability ordisrupts the integrity of (i.e., lyses or causes the formation of poresin) the membrane and/or cell wall of a cell (e.g., a somatic cell or amicrobial cell) to effect extraction or release of a biological analytenormally found in living cells.

“Detection system”, as used herein, refers to the components used todetect a biological analyte and includes enzymes, enzyme substrates,binding partners (e.g. antibodies or receptors), labels, dyes, andinstruments for detecting light absorbance or reflectance, fluorescence,and/or luminescence (e.g. bioluminescence or chemiluminescence).

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a housing that comprises “a”detection reagent can be interpreted to mean that the housing caninclude “one or more” detection reagents.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a cross-sectional view of one embodiment of a housingcomprising two receptacles and a cross-sectional view of a plungeradapted for use with the housing, which are both components of a samplepreparation and detection device according to the present disclosure.

FIG. 1B shows a cross-sectional view of the assembled device of FIG. 1Awith the plunger disposed in the housing in a first position andincluding a cell concentration agent in a upper receptacle of thehousing.

FIG. 1C shows a cross-sectional view of the device of FIG. 1B with theplunger disposed in the housing in a second position and including aliquid sample in the upper receptacle of the housing.

FIG. 1D shows a cross-sectional view of the device of FIG. 1C with theplunger in the first position and the cell concentration agent in alower receptacle of the housing.

FIG. 2A shows a cross-sectional view of one embodiment of a housingcomprising three receptacles separated by frangible seals and a sideview of a plunger adapted for use with the housing, which are bothcomponents of a sample preparation and detection device according to thepresent disclosure.

FIG. 2B shows a cross-sectional view of the housing of FIG. 2A with acap secured thereon and with a liquid sample disposed in an upperreceptacle of the housing.

FIG. 2C shows a cross-sectional view of the housing of FIG. 2B withoutthe cap and with a plunger disposed in a first position in the housing.

FIG. 2D shows a cross-sectional view of the device of FIG. 2C with theplunger disposed in a second position in the housing and the cellconcentration agent transferred to the lower receptacle of the housing.

FIG. 3A shows a front view of one embodiment of a sample preparation anddetection device comprising a housing and a valve, according to thepresent disclosure.

FIG. 3B shows a side view of the device of FIG. 3A.

FIG. 3C shows a cross-sectional view of the device of FIG. 3A with aliquid sample and a cell concentration agent disposed in a upperreceptacle of the housing and the valve in a first position.

FIG. 3D shows a cross-sectional view of the device of FIG. 3C with thevalve in a second position and the cell concentration agent transferredto the lower receptacle of the housing.

FIG. 4A shows a cross-sectional view of one embodiment of a housingcomprising two receptacles and a drain valve and a side view of aplunger, which are both components of a sample preparation and detectiondevice.

FIG. 4B shows a cross-sectional view of the assembled device of FIG. 4Awith the drain valve in an open configuration and the plunger disposedin a first position in the housing.

FIG. 4C shows a cross-sectional view of the assembled device of FIG. 4Bwith the plunger disposed in a second position in the housing and thecell concentration agent transferred to the second receptacle of thehousing.

FIG. 4D shows a cross-sectional view of the device of FIG. 4C, whereinthe plunger has punctured the frangible seals and transferred the cellconcentration agent to the lower receptacle.

FIG. 5A shows a cross-sectional view of one embodiment of a housing anda side view of a plunger, partially in section, which are bothcomponents of one embodiment of a sample preparation and detectiondevice according to the present disclosure.

FIG. 5B-5D show a cross-sectional views of the assembled device of FIG.5A with the plunger inserted to various depths into the housing.

FIG. 6A shows an exploded side view, partially in section, of the tip ofthe plunger of FIG. 5A.

FIG. 6B shows a side view, partially in section, of the assembled tip ofFIG. 6A.

FIG. 7A shows a cross-sectional view of one embodiment of a housing anda side view of a hollow plunger, partially in section, which are bothcomponents of one embodiment of a sample preparation and detectiondevice according to the present disclosure.

FIGS. 7B-7D show a cross-sectional views of the assembled device of FIG.7A with the plunger inserted to various depths into the housing.

FIG. 8A shows an exploded side view, partially in section, of the tip ofthe plunger of FIG. 7A.

FIG. 8B shows a side view, partially in section, of the assembled tip ofFIG. 8A.

FIG. 9 shows one embodiment of a cell concentration agent collectoraccording to the present disclosure.

FIG. 10 A shows a cross-sectional view of one embodiment of a housingand a side view of a plunger, partially in section, which are bothcomponents of one embodiment of a sample preparation and detectiondevice according to the present disclosure.

FIG. 10B shows a side view, partially in section, of the assembleddevice of FIG. 10A.

DETAILED DESCRIPTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The present invention generally relates to articles and methods fordetecting microorganisms in a sample. In certain preferred embodiments,the present invention relates to the detection of live microorganisms ina sample. Methods and devices for the concentration of cells from asample are described in U.S. Patent Application No. 61/141,900, filedDec. 31, 2008 entitled “SAMPLING DEVICES AND METHODS FOR CONCENTRATINGMICROORGANISMS” and U.S. Patent Application No. 61/141,813, filed Dec.31, 2008 entitled “METHODS, KITS AND SYSTEMS FOR PROCESSING SAMPLES”,each incorporated herein by reference in its entirety. The inventivedevices and methods disclosed herein provide increased sensitivity todetect small numbers of microorganisms present in a sample.

Biological analytes can be used to detect the presence of biologicalmaterial, such as live cells in a sample. Biological analytes can bedetected by various reactions (e.g., binding reactions, catalyticreactions, and the like) in which they can participate.

Chemiluminescent reactions can be used in various forms to detect cells,such as bacterial cells, in fluids and in processed materials. In someembodiments of the present disclosure, a chemiluminescent reaction basedon the reaction of adenosine triphosphate (ATP) with luciferin in thepresence of the enzyme luciferase to produce light provides the chemicalbasis for the generation of a signal to detect a biological analyte,ATP. Since ATP is present in all living cells, including all microbialcells, this method can provide a rapid assay to obtain a quantitative orsemiquantitative estimate of the number of living cells in a sample.Early discourses on the nature of the underlying reaction, the historyof its discovery, and its general area of applicability, are provided byE. N. Harvey (1957), A History of Luminescence: From the Earliest TimesUntil 1900, Amer. Phil. Soc., Philadelphia, Pa.; and W. D. McElroy andB. L. Strehler (1949), Arch. Biochem. Biophys. 22:420-433.

ATP detection is a reliable means to detect bacteria and other microbialspecies because all such species contain some ATP. Chemical bond energyfrom ATP is utilized in the bioluminescent reaction that occurs in thetails of the firefly Photinus pyralis. The biochemical components ofthis reaction can be isolated free of ATP and subsequently used todetect ATP in other sources. The mechanism of this fireflybioluminescence reaction has been well characterized (DeLuca, M., etal., 1979 Anal. Biochem. 95:194-198).

Samples and Sample Acquisition Devices:

Articles and methods of the present disclosure provide for the detectionof biological analytes in a sample. In some embodiments, the articlesand methods provide for the detection of biological analytes from livecells in a sample. In certain embodiments, the articles and methodsprovide for the detection of live microbial cells in a sample. Incertain preferred embodiments, the articles and methods provide for thedetection of live bacterial cells in a sample.

The term “sample” as used herein, is used in its broadest sense. Asample is a composition suspected of containing a biological analyte(e.g., ATP) that is analyzed using the invention. The biological analytemay be present in a cell (e.g. a bacterium) in the sample. While often asample is known to contain or suspected of containing a cell or apopulation of cells, optionally in a growth media, or a cell lysate, asample may also be a solid surface, (e.g., a swab, membrane, filter,particle), suspected of containing an attached cell or population ofcells. It is contemplated that for such a solid sample, an aqueoussample is made by contacting the solid with a liquid (e.g., an aqueoussolution) which can be mixed with cell concentration agents according tothe present invention.

Suitable samples include samples of solid materials (e.g., particulates,filters), semisolid materials (e.g., a gel, a liquid suspension ofsolids, or slurry), a liquid, or combinations thereof. Suitable samplesfurther include surface residues comprising solids, liquids, orcombinations thereof. Nonlimiting examples of surface residues includeresidues from environmental surfaces (e.g., floors, walls, ceilings,fomites, equipment, water, and water containers, air filters), foodsurfaces (e.g., vegetable, fruit, and meat surfaces), food processingsurfaces (e.g., food processing equipment and cutting boards), andclinical surfaces (e.g., tissue samples, skin and mucous membranes).Samples can also include mixtures such as crude or partially-refinedoil, gasoline, or paint.

The collection of sample materials, including surface residues, for thedetection of biological analytes is known in the art. Various sampleacquisition devices, including pipettes, spatulas, sponges, swabs andthe like have been described and can be used in the methods of thepresent invention.

Cell Concentration Agents:

Methods of the present disclosure include the use of cell concentrationagents to couple with cells that are present in a liquid sample. Thecell concentration agent is contacted for a period of time with a liquidsample suspected of containing cells. The cells can be coupled to thecell concentration agent either covalently, noncovalently (e.g., byhydrophobic or ionic interactions), or by a combination of covalent andnoncovalent coupling. After the cells have coupled to the cellconcentration agent, the cell concentration agent can be removed fromthe liquid sample by, for example, sedimentation, flocculation,centrifugation, filtration or any combination of the foregoing.

“Cell concentration agent” is used broadly to include materials (e.g.,particles, fibers) that can be suspended in a liquid and, thereby,capture and retain microorganisms that are present in the liquid.Although cell concentration agents can be collected by a filtrationprocess, they do not necessarily require a filtration process to capturethe microorganisms.

Certain cell concentration agents are known in the art and are suitablefor use in methods of the present disclosure. Nonlimiting examples ofsuitable cell concentration agents include hydroxyapatite (Berry et al.;Appl. Environ. Microbiol.; 63:4069-4074; 1997), magnetic beads (Oster etal., J. Magnetism and Magnetic Mat.; 225:145-150; 2001), ferrimagneticmineral, magnetite, chitosan, and affinity supports. The use ofcompositions including an immobilized-metal support material to captureor concentrate microorganisms from a sample is described in U.S. PatentApplication No. 60/913,812, filed on Apr. 25, 2007, and entitled“COMPOSITIONS, METHODS, AND DEVICES FOR ISOLATING BIOLOGICAL MATERIALS”,which is incorporated herein by reference in its entirety.

One exemplary type of concentration agents include diatomaceous earthand surface treated diatomaceous earth. Specific examples of suchconcentration agents can be found in commonly assigned U.S. PatentApplication No. 60/977,200, filed Oct. 3, 2007, and entitled“MICROORGANISMS CONCENTRATION PROCESS AND AGENT”; the disclosure ofwhich is incorporated herein by reference. When dispersed or suspendedin water systems, inorganic materials exhibit surface charges that arecharacteristic of the material and the pH of the water system. Thepotential across the material-water interface is called the “zetapotential,” which can be calculated from electrophoretic mobilities(that is, from the rates at which the particles of material travelbetween charged electrodes placed in the water system). In anembodiment, concentration agents can have zeta potentials that are atleast somewhat more positive than that of untreated diatomaceous earth,and the concentration agents can be surprisingly significantly moreeffective than untreated diatomaceous earth in concentratingmicroorganisms such as bacteria, the surfaces of which generally tend tobe negatively charged.

One exemplary type of concentration agent includes diatomaceous earth.Another exemplary type of concentration agent includes surface treateddiatomaceous earth. Exemplary surface treatment includes a surfacemodifier, such as titanium dioxide, fine-nanoscale gold or platinum, ora combination thereof. Such surface treatments can be surprisingly moreeffective than untreated diatomaceous earth in concentratingmicroorganisms. The surface treatment can also further include a metaloxide selected from ferric oxide, zinc oxide, aluminum oxide, and thelike, and combinations thereof. In an embodiment, ferric oxide isutilized. Although noble metals such as gold have been known to exhibitantimicrobial characteristics, the gold-containing concentration agentscan be effective not only in binding the microorganisms but also inleaving them viable for purposes of detection or assay.

Useful surface modifiers include fine-nanoscale gold; fine-nanoscaleplatinum; fine-nanoscale gold in combination with at least one metaloxide (for example, titanium dioxide, ferric oxide, or a combinationthereof); titanium dioxide; titanium dioxide in combination with atleast one other (that is, other than titanium dioxide) metal oxide; andthe like; and combinations thereof. In an embodiment, surface modifierssuch as fine-nanoscale gold; fine-nanoscale platinum; fine-nanoscalegold in combination with at least ferric oxide or titanium dioxide;titanium dioxide; titanium dioxide in combination with at least ferricoxide; or combinations thereof can be utilized.

In an embodiment surface modifiers such as the following can beutilized: fine-nanoscale gold; fine-nanoscale platinum; fine-nanoscalegold in combination with ferric oxide or titanium dioxide; titaniumdioxide; titanium dioxide in combination with ferric oxide; andcombinations thereof. In an embodiment, fine-nanoscale gold;fine-nanoscale gold in combination with ferric oxide or titaniumdioxide; titanium dioxide in combination with ferric oxide; andcombinations thereof can be utilized. Fine-nanoscale gold,fine-nanoscale gold in combination with ferric oxide or titaniumdioxide, and combinations thereof can also be utilized in an embodiment.

Another exemplary type of concentration agent includes gamma-FeO(OH)(also known as lepidocrocite). Specific examples of such concentrationagents can be found in commonly assigned U.S. Patent Application No.60/977,188, filed Oct. 3, 2007, and entitled “MICROORGANISMCONCENTRATION PROCESS”; the disclosure of which is incorporated hereinby reference. Such concentration agents have been found to besurprisingly more effective than other iron-containing concentrationagents in capturing gram-negative bacteria, which can be of greatconcern in regard to food- and water-borne illnesses and human bacterialinfections. The concentration agents can further include (in addition togamma-FeO(OH)) other components (for example, boehmite (α-AlO(OH)),clays, iron oxides, and silicon oxides). In embodiments where such othercomponents are included, they generally do not significantly interferewith the intimate contact of the sample and the concentration agent.

Gamma-FeO(OH) is known and can be chemically synthesized by knownmethods (for example, by oxidation of ferrous hydroxide at neutral orslightly acidic pHs, as described for purposes of magnetic tapeproduction in U.S. Pat. No. 4,729,846 (Matsui et al.), the descriptionof which is incorporated herein by reference). Gamma-FeO(OH) is alsocommercially available (for example, from Alfa Aesar, A Johnson MattheyCompany, Ward Hill, Mass., and from Sigma-Aldrich Corporation, St.Louis, Mo.).

In an embodiment that utilized gamma-FeO(OH) as a concentration agent,the gamma-FeO(OH) is generally in the form of microparticles. In anembodiment, it is in the form of microparticles having particle sizes(largest dimension) in the range of about 3 micrometers (in otherembodiments, about 5 micrometers; or about 10 micrometers) to about 100micrometers (in other embodiments, about 80 micrometers; or about 50micrometers; or about 35 micrometers; where any lower limit can bepaired with any upper limit of the range). In an embodiment, theparticles are agglomerates of smaller particles. The particles caninclude crystallites that are less than about 1 micrometer in size (inan embodiment, less than about 0.5 micrometer in size). The crystallitescan be present as acicular crystallites, as raft-like structurescomprising acicular crystallites, or as combinations of the acicularcrystallites and raft-like structures.

In an embodiment, the concentration agents have a surface area asmeasured by the BET (Brunauer-Emmett-Teller) method (calculation of thesurface area of solids by physical adsorption of nitrogen gas molecules)that is greater than about 25 square meters per gram (m²/g); in anembodiment greater than about 50 m²/g; and in another embodiment greaterthan about 75 m²/g.

An agglomerated form of the particles can provide the adsorptivecapabilities of fine particle systems without the handling and otherhazards often associated with fine particles. In addition, suchagglomerate particles can settle readily in fluid and thus can providerapid separation of microorganisms from a fluid phase (as well asallowing low back pressure when filtered).

Another exemplary type of concentration agents includes metal silicates.Specific examples of such concentration agents can be found in commonlyassigned U.S. Patent Application No. 60/977,180, filed Oct. 3, 2007, andentitled “MICROORGANISM CONCENTRATION PROCESS”; the disclosure of whichis incorporated herein by reference. Exemplary metal silicates can havea surface composition having a metal atom to silicon atom ratio of lessthan or equal to about 0.5 (in an embodiment, less than or equal toabout 0.4; in another embodiment, less than or equal to about 0.3; inyet another embodiment, less than or equal to about 0.2), as determinedby X-ray photoelectron spectroscopy (XPS). In an embodiment, the surfacecomposition also includes at least about 10 average atomic percentcarbon (in an embodiment, at least about 12 average atomic percentcarbon; in yet another embodiment at least about 14 average atomicpercent carbon), as determined by X-ray photoelectron spectroscopy(XPS). XPS is a technique that can determine the elemental compositionof the outermost approximately 3 to 10 nanometers (nm) of a samplesurface and that is sensitive to all elements in the periodic tableexcept hydrogen and helium. XPS is a quantitative technique withdetection limits for most elements in the 0.1 to 1 atomic percentconcentration range. Exemplary surface composition assessment conditionsfor XPS can include a take-off angle of 90 degrees measured with respectto the sample surface with a solid angle of acceptance of ±10 degrees.

When dispersed or suspended in water systems, inorganic materials suchas metal silicates exhibit surface charges that are characteristic ofthe material and the pH of the water system. The potential across thematerial-water interface is called the “zeta potential,” which can becalculated from electrophoretic mobilities (that is, from the rates atwhich the particles of material travel between charged electrodes placedin the water system). Exemplary concentration agents can have zetapotentials that are more negative than that of, for example, a commonmetal silicate such as ordinary talc. Yet the concentration agents aresurprisingly more effective than talc in concentrating microorganismssuch as bacteria, the surfaces of which generally tend to be negativelycharged. In an embodiment, the concentration agents have a negative zetapotential at a pH of about 7 (in an embodiment, a Smoluchowski zetapotential in the range of about −9 millivolts to about −25 millivolts ata pH of about 7; in another embodiment, a Smoluchowski zeta potential inthe range of about −10 millivolts to about −20 millivolts at a pH ofabout 7; in yet another embodiment a Smoluchowski zeta potential in therange of about −11 millivolts to about −15 millivolts at a pH of about7).

Useful metal silicates include, but are not limited to, amorphoussilicates of metals such as magnesium, calcium, zinc, aluminum, iron,titanium, and the like, and combinations thereof. In an embodiment,magnesium, zinc, iron, titanium, or combinations thereof can beutilized. In yet another embodiment, magnesium is utilized. In anembodiment, amorphous metal silicates in at least partially fusedparticulate form can be utilized. In an embodiment, amorphous,spheroidized metal silicates can be utilized. In yet another embodiment,amorphous, spheroidized magnesium silicate can be utilized. Metalsilicates are known and can be chemically synthesized by known methodsor obtained through the mining and processing of raw ores that arenaturally-occurring.

Some amorphous metal silicates are commercially available. For example,amorphous, spheroidized magnesium silicate is commercially available foruse in cosmetic formulations (for example, as 3M Cosmetic MicrospheresCM-111, available from 3M Company, St. Paul, Minn.).

In addition to amorphous metal silicates, the concentration agents canalso include other materials including oxides of metals (for example,iron or titanium), crystalline metal silicates, other crystallinematerials, and the like, provided that the concentration agents have theabove-described surface compositions. In an embodiment, a concentrationagent contains essentially no crystalline silica.

The concentration agents can be used in any form that is amenable tosample contact and microorganism capture. In an embodiment, theconcentration agents are used in particulate form. In an embodiment, theconcentration agent is in the form of microparticles. In an embodiment,the concentration agent is in the form of microparticles having aparticle size in the range of about 1 micrometer (in an embodiment,about 2 micrometers) to about 100 micrometers (in an embodiment, about50 micrometers; in another embodiment, about 25 micrometers; in yetanother embodiment about 15 micrometers; where any lower limit can bepaired with any upper limit of the range).

Microbial concentration or capture using concentration agents isgenerally not specific to any particular strain, species, or type ofmicroorganism and therefore provides for the concentration of a generalpopulation of microorganisms in a sample. Specific strains ofmicroorganisms can then be detected from among the capturedmicroorganism population using any known detection method withstrain-specific probes. Thus, the concentration agents can be used forthe detection of microbial contaminants or pathogens (particularlyfood-borne pathogens such as bacteria) in clinical, food, environmental,or other samples.

In carrying out the process of the invention, the concentration agentscan be used in any form that is amenable to sample contact andmicroorganism capture (for example, in particulate form or applied to asupport such as a dipstick, film, filter, tube, well, plate, beads,membrane, or channel of a microfluidic device, or the like). Preferably,the concentration agents are used in particulate form.

Optionally, the cell concentration agent may comprise a binding partner(e.g., an antibody, an antibody fragment, an antigen-binding domain, alectin (e.g., Concanavalin A), a receptor, a phage receptor, or thelike), which can couple to a microorganism. The coupling can be director indirect. The coupling can be selective for certain microorganismtypes or it can be nonselective.

The amount of concentration agent used to capture microorganisms from asample can depend at least in part on the type of concentration agentutilized, the sample size, the receptacle type and size, sample mixing,the particular application, other factors not specifically discussedherein, or a combination thereof. The capture efficiency (the percent ofmicroorganisms in the sample bound to concentration agent) can generallybe increased by allowing increased time for the microorganism to come incontact with the concentration agent. The capture efficiency can also beincreased by having a higher concentration of concentration agent, whichdecreases the mean diffusion distance a microorganism must travel to becaptured, leading to a shorter incubation time. Therefore, as agenerality, the more concentration agent added, the shorter incubationtime necessary to capture the same amount of microorganisms.

In an embodiment, an appropriate amount of concentration agent can varygiven the time necessary to wait for the microorganisms to be bound tothe concentration agent (referred to as “capture time”). For example,for a capture time of 1 minute, 1000 mg of concentration agent per 10 mLof sample could be appropriate; for a capture time of 10 minutes, 100 mgof concentration agent per 10 mL of sample could be appropriate; and fora capture time of 60 minutes, 10 mg of concentration agent per 10 mL ofsample could be appropriate. In an embodiment, from about 1 mg to about100 mg of concentration agent per 10 mL of sample can be utilized. In anembodiment, from about 1 mg to about 50 mg of concentration agent per 10mL of sample can be utilized. In an embodiment, from about 10 mg toabout 25 mg of concentration agent per 10 mL of sample can be utilized.In an embodiment utilizing a metal silicate concentration agent forexample, about 10 mg of a metal silicate concentration agent per 10 mLof sample can be utilized. In an embodiment utilizing a metal silicateconcentration agent for example, about 25 mg of a metal silicateconcentration agent per 10 mL of sample can be utilized.

Detection Devices:

The present disclosure provides devices that can be used to detectmicroorganisms in a sample. The devices can include a housing comprisingat least two receptacles with a passageway therebetween, an optionalcell concentration agent disposed in an upper receptacle of the housing,a means for isolating at least two receptacles in the housing, and meansfor transferring the cell concentration agent from the upper receptacleto a lower receptacle of the housing. In some embodiments, the housingcan include the means (e.g. a frangible seal) for isolating the tworeceptacles. In some embodiments, the housing can include the means(e.g., a valve) for transferring the cell concentration agent from theupper receptacle to the lower receptacle of the housing. In someembodiments, the devices further can include a reagent for detectingmicroorganisms. In certain embodiments, the devices further can includea hydrogel comprising a cell extractant. The cell extractant canfacilitate the detection of a biological analyte from the microorganism.

Turning now to the drawings, FIG. 1A shows a cross-sectional view of thecomponents of one embodiment of a detection device 100 according to thepresent disclosure. The detection device components comprise a housing110 and a plunger 150. The housing 110 includes an upper part 112adjacent a lower part 114. The upper part 112 and lower part 114 can beformed separately from polymeric material, such as polyethylene orpolypropylene, by processes that are well-known in the art such as, forexample, molding. The parts can be dimensioned such that they can bepress-fit together to provide a substantially liquid-tight coupling or,alternatively, they can be coupled together by means that are known inthe art (e.g., by an adhesive, sonic welding, or the like).Alternatively, the housing could be formed as a single unit by processesthat are known in the art, such as extruding a hollow body, molding thepassageway, and sealing the bottom of the housing with a processinvolving heat, for example. In other embodiments, an insert part,comprising the narrow passageway, could be placed into a unitary housingto form the upper and lower receptacles (120 and 124, respectively).

At the end of the upper part 112 distal the lower part 114, is anopening 113 that is dimensioned to receive the plunger 150. At theopposite end of the upper part 112 is a passageway 116 that opens intothe lower part 114 of the housing 110. In the illustrated embodiment,the passageway 116, which has a cross-sectional area that is smallerthan the cross-sectional area of the upper receptacle 120, is shown asan inward extension of the wall that forms the upper part 112.Alternatively, the passageway 116 could be formed by an insert that fitsinside the wall of the upper part 112 adjacent the lower part 114 of thehousing 110 (not shown). The insert could form the passageway 116adjacent the lower part 114 of the housing 110. The relative proportionsof the upper part 112, lower part 114, and passageway 116 in FIG. 1A aremerely illustrative and can be adapted, as necessary to accommodatevarious parameters, such as sample volume and/or instrument limitations.

The plunger 150 comprises a shaft 151 with a handle 152 at one end and aplurality of seals (first lower seal 156 and second lower seal 157) atthe opposite end. Optionally, the plunger 150 can comprise one or moreupper seals 154 and/or an index mark 153. The relative distances betweenthe handle 152, first lower seal 156 and second lower seal 157 aredescribed below. Also shown in FIG. 1A is optional detection reagent 165and optional hydrogel 162.

“Detection reagent” is used herein in its broadest sense. A detectionreagent is a reagent that can be used in a reaction to detect abiological analyte. Nonlimiting examples of detection reactions includeinteraction between binding partners (e.g., antigen-antibody,receptor-ligand, probe-target, and hybridization binding interactions)and/or catalytic reactions (e.g., enzyme-mediated reactions such as, forexample, fluorogenic reactions, chromogenic reactions, lumigenicreactions, or polymerization reactions). Detection reagents mayparticipate (e.g., as a binding partner, an enzyme, an enzyme substrate,or an indicator) in the detection reaction and/or may facilitate (e.g.,as a buffer, a cofactor, or a component of a coupled reaction) adetection reaction. Exemplary detection reagents include enzymes,including, for example, luciferase, adenylate kinase, peroxidase,alkaline phosphates, apyrase, and the like; enzyme substrates,including, for example, luciferin, methylumbelliferyl phosphate,o-nitrophenylphosphate, p-nitrophenylphosphate, and5-bromo-4-choloro-3-indoxyl-phosphate; buffers, including, for example,phosphate buffer, TRIS buffer, and HEPES buffer; and cofactors,including, for example, FADH, NADH, coenzyme A, and the like.

Detection reagents can be included in the housing 110 in variousconfigurations. For example, the detection reagent 165 can comprise adried or partially-dried coating, as shown in FIG. 1A. Suitablealternative configurations (not shown) for the detection reagent 165 arewell known in the art and include, for example, liquid reagents(optionally, in a frangible compartment, such as an ampoule), powders,gels, tablets, lyophilized reagents, coated films, cakes, and dried-downreagents.

FIG. 1B shows a cross-sectional view of a detection device 100comprising the housing 110 with the plunger 150 of FIG. 1A. This drawingillustrates a configuration in which the device 100 can be stored beforeuse. The plunger 150 is fully-inserted in the housing 110. In thisposition, the lower edge of the handle 152 blocks the opening 113 of theupper part 112 of the housing 110, thereby preventing material fromentering or exiting the housing 110. Optional upper seals 154 can alsoserve to prevent materials from entering or exiting the housing 110. Theupper seals 154 are dimensioned to contact the inner surface of the wallof the upper part 112 of the housing 110 and are made of a suitablematerial (e.g., poly propylene, butyl rubber) to form a barrier,preferably a liquid-resistant barrier.

When the plunger 150 is in the position shown in FIG. 1B, the firstlower seal 156 blocks the passageway 116, thereby isolating the upperreceptacle 120 from the lower receptacle 124 of the housing 110. Whenthe plunger 150 is in the position shown in FIG. 1B, a portion of theplunger 150 which includes the second lower seal 157 extends into thelower receptacle 124 and does not contact the walls of lower part 114 ofthe housing 110. The first lower seal 156 and second lower seal 157 aredimensioned to contact the walls of the passageway 116 and are made of asuitable material (e.g., poly propylene, butyl rubber) to form abarrier, preferably a liquid-resistant barrier in the passageway 116between the upper receptacle 120 and the lower receptacle 124. Alsoshown in FIG. 1B is an optional concentration agent 130, located in theupper receptacle 120.

FIG. 1C shows a cross-sectional view of the device 100 of FIG. 1B withthe plunger 150 in a second position. This plunger 150 position can beused, for example, to load a sample into the housing 110. The plunger150 can be grasped by the handle 152 and withdrawn until the secondlower seal 157 is proximate the upper end of the passageway 116. Theoptional index mark 153 on the plunger shaft 151 can be used (e.g., whenit is aligned with the opening 113) to indicate the proper location ofthe plunger 150 to attain this position. FIG. 1C further comprises aliquid sample 140 that is contacting the concentration agent 130 in theupper receptacle 120. During use, the device 100 can be vortexed orvibrated, for example, to mix the concentration agent 130 and the liquidsample 140. After a period of time, the concentration agent 130 cansettle to the bottom of the upper receptacle 120, as shown in FIG. 1C.In some embodiments, an optional taper region 118 is located adjacentthe passageway 116. The taper region 118 can be formed from the samematerial and/or process as the upper part 112 of the housing 110 and/orthe passageway 116. In use, the taper region 118 can direct toward thepassageway 116 liquid-suspended particles (e.g., cell concentrationagent 130) that are sedimenting toward the passageway 116 within thehousing 110.

FIG. 1D shows a cross-sectional view of the device 100 of FIG. 1C withthe plunger 150 returned to the first position shown in FIG. 1B. Thelower edge of the handle 152 is proximate the opening 113 and a portion142 of the liquid sample, containing the concentration agent 130 istransferred to the lower receptacle 124, where the portion 142 caninteract with a detection reagent 165 (shown in FIG. 1A), if present.Non-limiting examples of interactions between the portion 142 and thedetection reagent 165 include dissolution and/or suspension of thedetection reagent, binding interactions between the detection reagentand a biological analyte present in the portion, and/or a catalyticreaction. FIG. 1D also shows the portion 142 of the liquid samplecontacting the hydrogel 162 in the lower receptacle 124, which canresult in the release of a cell extractant from the hydrogel 162.

In the embodiment illustrated in FIG. 1, the means for isolating theupper receptacle 120 from the lower receptacle 124 comprises the firstlower seal 156 and/or second lower seal 157 of the plunger 150 incombination with the passageway 116. In the embodiment illustrated inFIG. 1, the means for transferring the concentration agent 130 from theupper receptacle 120 to the lower receptacle 124 includes the passageway116 and the first lower seal 156 and second lower seal 157 of theplunger 150.

FIG. 2A shows a cross-sectional view of a plunger 250 and apartially-exploded cross-sectional view of a housing 210, which arecomponents of one embodiment of a detection device 200 according to thepresent invention. The housing 210 includes an upper part 212 adjacent alower part 214. The upper part 212 and lower part 214 can be formed asdescribed above.

At the end of the upper part 212 distal the lower part 214, is anopening 213 that is dimensioned to receive the plunger 250. At theopposite end of the upper part 212 is a passageway 216, as describedabove. Adjacent the passageway 216 is an optional taper region 218, asdescribed herein. Frangible seals 260 a and 260 b divide the housinginto the upper receptacle 220, lower receptacle 224, and thirdreceptacle 226. Frangible seals 260 a and 260 b are preferably made froma water-resistant material (e.g., a thin polymeric film, apolymer-coated paper, a thin foil) and can be secured to the walls ofthe housing 210 using materials and/or processes that are known in theart (e.g., an adhesive, heat-sealing, ultrasonic welding) to form awater-resistant frangible barrier.

Located in the third receptacle 226 is a hydrogel 262 comprising a cellextractant. Suitable hydrogels comprising a cell extractant aredescribed in U.S. Patent Application No. 61/101,546, filed on Sep. 30,2008, and entitled “BIODETECTION ARTICLES”, which is incorporated hereinby reference in its entirety.

The relative proportions of the three receptacles in FIG. 2A are merelyillustrative and can be adapted, as necessary to accommodate variousparameters, such as sample volume and/or instrument limitations. Alsoshown in FIG. 2A are an optional concentration agent 230, optionaldetection reagent 265 as described herein and optional removable cap278. Cap 278 can be made from, for example, a polymeric material (e.g.,polyethylene, polypropylene) using processes known in the art (e.g.,molding) and can be dimensioned to form a liquid-resistant cover for thehousing 210.

The plunger 250 comprises a shaft 251 with a handle 252 at one end andthe lower seal 256 and piercing end 259 at the opposite end. Preferably,the lower seal 256 dimensioned to contact the walls of the passageway216 and is made of a suitable material (e.g., poly propylene, butylrubber) to form a barrier, preferably a liquid-resistant barrier, in thepassageway 216. Optionally, the plunger 250 can comprise one or moreupper seals 254 as described above. The relative distances between thehandle 252, lower seal 256 and the piercing end 259 are described below.

FIG. 2B shows a cross-sectional view of the device 200 of FIG. 2A. Inthis view, the housing 210 further comprises a liquid sample 240 in theupper receptacle 220. The cap 278 is firmly seated on the housing 210and, thus, the liquid sample 240 can be mixed with the cellconcentration agent 230 by processes that are known in the art such as,for example, vortexing, vibrating, shaking, or inverting the housing210. After mixing, the cell concentration agent 230 can be allowed tosettle onto the frangible seal 260 a in the passageway 216.

FIG. 2C shows a cross-sectional view of the device 200 comprising thehousing 210 of FIG. 2B with a plunger 250 partially inserted therein. Inthis position, the lower seal 256 of the plunger 250 contacts the wallsof the passageway 216, thereby isolating in the passageway 216 at leasta portion 242 from the rest of the liquid sample 240. Also isolated inthe passageway 216 is the cell concentration agent 230.

FIG. 2D shows a cross-sectional view of the device 200 of FIG. 2C withthe plunger 250 fully inserted therein. The lower seal 256 of theplunger 250 contacts the walls of the passageway 216 and the piercingend 259 has punctured frangible seals 260 a and 260 b, therebytransferring the portion 242 of the liquid sample, the cellconcentration agent 230, and the hydrogel 262 into the lower receptacle224, where the portion 242 can interact with optional detection reagent265 (shown in FIG. 2A), if present. Non-limiting examples ofinteractions between the portion 242 and the detection reagent 265include dissolution and/or suspension of the detection reagent, bindinginteractions between the detection reagent and a biological analytepresent in the portion, and/or a catalytic reaction.

In the illustrated embodiment of FIG. 2, the means for isolating theupper receptacle 220 from the lower receptacle 224 includes thefrangible seals 260 a and 260 b. Means for isolating the upperreceptacle 220 from the lower receptacle 224 can also include the lowerseal 256 of the plunger 250 in combination with the passageway 216. Inthe illustrated embodiment of FIG. 2, the means for transferring thecell concentration agent 230 from the upper receptacle 220 to the lowerreceptacle 224 includes the piercing end 259 and lower seal 256 of theplunger 250 and the passageway 216.

FIG. 3A shows a front view of one embodiment of a detection device 300according to the present disclosure. The device 300 includes a housing310 and an optional cap 378. The housing 310 can be constructed asdescribed above with an upper part 312, a passageway 316, and a lowerpart 314. The optional cap 378 can be constructed as described above.The device 300 also includes a dead-end valve 370 with a valve actuator372, which is shown in a first position in FIG. 3A. FIG. 3B shows a sideview of the device 300 and valve actuator 372 of FIG. 3A.

FIG. 3C show a cross-sectional view of the device 300 shown in FIG. 3A.The device 300 comprises a cap 378 and a housing 310. The housing 310includes an upper part 312 and lower part 314. The upper part 312includes a passageway 316 in which a dead-end valve 370 is positioned.The dead-end valve 370 includes a valve cavity 374 which, when the valveis in this first position, is in fluid communication with the upperreceptacle 320. The valve cavity 374 includes an optional cellconcentration agent 330, which contacts a liquid sample 340 in the upperreceptacle 320. The lower receptacle 324 contains an optional hydrogel362 and/or optional detection reagent 365, both as described herein.Also shown in FIG. 3C is optional taper region 318, as described herein.

FIG. 3D shows a cross-sectional view of the device 300 from FIG. 3C withthe valve 370 in a second position. When the valve 370 is in the secondposition, a portion 342 of the liquid sample, containing the cellconcentration agent 330, is isolated and transferred to the lowerreceptacle 324 where the portion 342 can contact the hydrogel 362, ifpresent, and can interact with the detection reagent 365, if present, asdescribed herein.

It is recognized that the dimensions of the valve cavity 374 canconstitute a known predetermined volume and that, as such, the valve 370can be used one or more times to transfer a predetermined amount of theliquid sample 340 from the upper receptacle 320 to the lower receptacle324. Furthermore, it is recognized that, after the portion 342 of theliquid sample has been transferred from the upper receptacle 320 to thelower receptacle 324, the remainder of the liquid sample 340 in theupper receptacle 320 could be discarded and a different material (e.g.,a diluent, a buffer, a liquid and/or powder reagent) can be placed intothe upper receptacle 320 and a predetermined amount could subsequentlybe transferred to the lower receptacle 324 using the valve 370 (notshown).

In the illustrated embodiment of FIG. 3, the means for isolating theupper receptacle 320 and lower receptacle 324 of the housing includesthe valve 370. In the illustrated embodiment of FIG. 3, the means fortransferring the cell concentration agent 330 from the upper receptacle320 to the lower receptacle 324 includes the passageway 316 and thevalve 370.

FIG. 4A shows a side view of a plunger 450 and a cross-sectional view ofa housing 410, both of which are components of a detection device 400according to the present disclosure. The plunger comprises a shaft 451,optional O-ring 455, and piercing end 459. The O-ring can be made of aconformable material (e.g., butyl rubber) to provide a liquid-tight sealwith the housing 410. The housing 410 can be constructed as describedabove with an upper part 412 and a lower part 414. The optional cap 478can be constructed as described above. Frangible seals 460 divide thehousing 410 into three receptacles, an upper receptacle 420, lowerreceptacle 424, and third receptacle 426. In this illustration,frangible seals 460 are located at the end of the upper receptacle 420that is proximate the lower receptacle 424. The space between thefrangible seals 460 defines a third receptacle 426. Located in the thirdreceptacle 426 is a hydrogel 462 comprising a cell extractant. Analternative construction (not shown) may have only one frangible seal460 proximate the lower receptacle 424, with the hydrogel 462 located inthe lower receptacle 424, as shown in FIG. 3C.

Located in the upper receptacle 420 proximate frangible seals 460 is adrain valve 480 with the valve gate 482, which is shown in the closedposition. Also located in the upper receptacle 420 is the liquid sample440 and the optional cell concentration agent 430. An optional detectionreagent 465 is shown in the lower receptacle 424.

FIG. 4B shows a cross-sectional view of an assembled detection device400 comprising the housing 410 and plunger 450 of FIG. 4A. The cellconcentration agent 430 is settled to the bottom of the upper receptacle420. The valve gate 482 of the drain valve 480 is in the open positionand, as force is applied (e.g., by pressure from finger or hand) in thedirection shown by the arrow, the clarified liquid sample 445 isexpelled out of the drain valve 480. Also shown in FIG. 4B is detectionreagent 465 coated on the wall of the lower receptacle 424.

FIG. 4C shows a cross-sectional view of the detection device 400 of FIG.4B. In this view, the O-ring 455 and piercing end 459 of the plunger 450are inserted in the housing 410 on the side of the drain valve 480proximate the nearest frangible seal 460. In this position, the plunger450 traps a portion 442 of the liquid sample comprising the cellconcentration agent 430 between the plunger 450 and the nearestfrangible seal 460.

FIG. 4D shows a cross-sectional view of the detection device 400 of FIG.4C. In this view, the piercing end 459 of the plunger 450 has puncturedboth frangible seals 460 and the portion 442 of the liquid sample hastransferred to the lower receptacle 424, where it has dissolved thedetection reagent (shown in FIG. 4C) and the portion 442 is in contactwith the hydrogel 462 comprising a cell extractant.

FIG. 5A shows a cross-sectional view of a plunger 550 and a housing 510,both of which are components of a detection device 500. The plunger 550comprises a shaft 551 with an optional handle 552 and a tip 590. In anyembodiment, the handle 552 further may comprise an optional O-ring 555.

The housing 510 can be constructed as described above, with an upperpart 512 and a lower part 514. Frangible seals 560 a and 560 b dividethe housing 510 into three receptacles, an upper receptacle 520, lowerreceptacle 524, and third receptacle 526. In this illustration,frangible seals 560 a and 560 b are located at the end of the upperreceptacle 520 that is proximate the lower receptacle 524. The spacebetween the frangible seals 560 a and 560 b defines the third receptacle526. Located in the third receptacle 526 is hydrogel 562, whichcomprises a cell extractant as described herein. In the illustratedembodiment, the lower receptacle 524 comprises an optional detectionreagent 565. An alternative construction (not shown) may have only onefrangible seal proximate the lower receptacle, with the hydrogel locatedin the lower receptacle, as shown in FIG. 3C.

The handle 552 can be made, using processes well known in the art, froma variety of materials including, for example, plastic, wood, metal, andcombinations thereof. The optional O-ring 555 is disposed in a notch 554in the handle 552. The handle 552 may be shaped and dimensioned suchthat at least a portion of the handle 552 can be inserted into thehousing 510 when the plunger 550 is fully inserted in the housing 510.In one embodiment, the handle 552 further includes a rim 554 thatengages the opening of the housing 510 to prevent the handle 552 frombeing fully inserted into the housing 510.

The shaft 551 of the plunger 550 can be made from a variety of materialsincluding, for example, plastic, wood, metal, and combinations thereof.One end of the shaft 551 is coupled to the handle 552 by press-fittinginto a recessed portion (as shown in FIG. 5), by ultrasonic welding, orby using an adhesive, for example. The other end of the shaft 551 iscoupled to the tip 590 by press-fitting, by ultrasonic welding, or byusing an adhesive, for example.

Detail of the tip 590 of the plunger 550 is shown in FIGS. 6A and 6B.

FIG. 6A shows a partially exploded side view, partially in section, ofthe tip 590 of FIG. 5A. The tip 690 comprises a body 691, a one-wayvalve 697, and a filter 696.

The body 691 includes a first end 691 a, a second end 691 b, and aconduit 692 running through the body 691 from the first end 691 a to thesecond end 691 b. At the first end 691 a, the conduit 692 is sealed bythe shaft 651 of the plunger. At the second end 691 b, the conduit 692opens into a recessed opening 694. Two drain channels 695 run from thefirst end 691 a of the body 691 to the conduit 692. Thus, the drainchannels are fluidically connected to the conduit 692 and the recessedopening 694. In one embodiment (not shown), the tip 690 may compriseonly one drain channel 695. Advantageously, a plurality of drainchannels 695 may provide less back-pressure and, thus, a higher rate offluid transport through the tip 690.

The body 691 may be fabricated from plastic (e.g., polypropylene,polyethylene, polytetrafluoroethylene) by molding, for example. The body691 is shaped and dimensioned to fit in a housing (e.g., housing 510 ofFIG. 5A). In any embodiment, the body 691 or the O-ring 686 can form asubstantially liquid-tight seal with the walls of a housing when thebody 691 is inserted into the housing. In any embodiment that includesan O-ring, 686, the O-ring 686 may function both to form a liquid-tightseal and to wipe particulate material (e.g., cell concentration agents)off the wall of the housing as the O-ring 686 is moved in relation tothe wall of the housing. The shaft 651 may be coupled to the conduit 692by means that are known in the art (e.g., by an adhesive, by press-fit).The optional O-ring 686 is disposed in a notch 689 in the body 691.

The tip 690 further comprises a filter 696. The filter 696 is coupled tothe body 691. In the illustrated embodiment, the filter 696 is formedfrom a porous material, which can be press-fit and/or adhesively coupledto the recessed opening 694. In some embodiments, the porous materialcan be semi-rigid porous material (e.g., POREX filtration medium soldunder the part number X6854 by Porex Corporation, Fairburn, Ga.). Thefilter 696 may be configured with a relatively angular or pointed end,such that the end can facilitate the penetration of a frangible seal. Inalternative embodiments (not shown), the filter may comprise a membranefilter that is coupled to the body. When coupled to the body, themembrane filter is part of a fluid path that includes the conduit and adrain channel.

In some embodiments, the porosity of the filter 696 may be selected suchthat the filter 696 prevents only the passage of relatively largeparticles (e.g., >1 μm, >5 μm, or >10 μm,) through it. Relatively largeparticles may include, for example, cell concentration agents asdescribed herein. In these embodiments, microorganisms such as bacteria,yeast, and/or filamentous fungi (mold) may pass through the filter 696.

In some embodiments, the porosity of the filter 696 may be selected suchthat the filter 696 prevents only the passage of relatively smallparticles (e.g., <1 μm, <0.45 μm, <0.2 μm,) through it. In theseembodiments, microorganisms such as bacteria, yeast, and/or filamentousfungi (mold) may be retained by the filter 696.

The tip 690 further comprises a one-way valve 697 disposed in therecessed opening 694 between the filter 696 and the conduit 692. Alsoshown is an optional retaining washer 698 that serves to hold theone-way valve 697 in position. The one-way valve 697 may be constructedfrom plastic (e.g., polypropylene, polyethylene, polyester) or rubber,for example, and may be configured as a duck-bill valve, for example. Inuse, the one-way valve 697 substantially prevents the flow of liquidthat has passed through the filter 696 from returning through the filter696 in the opposite direction.

FIG. 6B shows a side view, partially in section of the assembled tip 690of FIG. 6A. The one-way valve 697, optional retaining washer 698, andfilter 696 are disposed in the recessed opening and are in fluidicconnection with the conduit 692 and the drain channels 695. The shaft651 is coupled to the body 691 of the tip 690.

Referring back to FIG. 5A, the detection device 500 comprising thehousing 510 and plunger 550 is used in a method to detect microorganismsand, in particular, live microorganisms.

In use, a liquid sample is transferred into the upper receptacle 520 ofthe housing 510, where it is allowed to contact a cell concentrationagent 530. After adding the liquid sample 540 to the housing 510, thetip of the plunger 550 is inserted into the housing 510 and urged (e.g.,manually or mechanically) toward the lower receptacle 524 of the housing510, as shown in FIG. 5B. As the tip 591 of the plunger 550 contacts theliquid sample 540, the liquid passes through the tip 590 and back intothe housing 510, as shown in FIG. 5B. This process retains the cellconcentration agent 530 and, in some embodiments, free microorganisms ina portion 542 of the liquid sample proximate the third receptacle 526.

As the tip 590 of the plunger 550 penetrates the frangible seal 560 a,not shown, the portion 542 of the liquid sample containing the cellconcentration agent 530 contacts the hydrogel 562. Further movement ofthe plunger 550 (as shown in FIG. 5D) causes penetration of thefrangible seal 560 b, which causes the portion 542 of the liquid sampleand the hydrogel 562 to transfer to the lower receptacle 524, where theycontact the detection reagent 565.

FIG. 7A shows a cross-sectional side view of another embodiment of adetection device 700 according to the present disclosure. The detectiondevice 700 comprises a plunger 750 and a housing 610.

The housing 710 can be constructed as described above with an upper part712 and a lower part 714. Frangible seals 760 a and 760 b divide thehousing 710 into three receptacles, an upper receptacle 720, lowerreceptacle 724, and third receptacle 726. In this illustration,frangible seals 760 a and 760 b are located at the end of the upperreceptacle 720 that is proximate the lower receptacle 724. The spacebetween the frangible seals 760 a and 760 b defines a third receptacle726. Located in the third receptacle 726 is a hydrogel 762 comprising acell extractant. In the illustrated embodiment, the lower receptacle 724comprises an optional detection reagent 765. An alternative construction(not shown) may have only one frangible seal 760 proximate the lowerreceptacle 724, with the hydrogel 762 located in the lower receptacle724, as shown in FIG. 3C.

The plunger 750 comprises a shaft 751 coupled to a handle 752 and a tip790. In this embodiment, the shaft 751 is hollow and the handlecomprises a vent 748 to equalize the pressure between the interior andexterior of the shaft 751. The plunger further comprises an optionaldrain tube 753. The drain tube 753 receives liquid filtrate from the tip790 and distributes the filtrate to the interior of the shaft 751. Byfunctioning as an overflow valve, the drain tube 753 also reduces thevolume of filtrate that can flow back through the tip 790 in the reversedirection.

Detail of the tip 790 of the plunger 750 is shown in FIG. 8.

FIG. 8A shows a partially exploded side view, partially in section, ofthe tip 790 of FIG. 7A. The tip 890 comprises a body 891, an optionalone-way valve 897, and a filter 896. Also show in FIG. 8A is a portionof the plunger 850 comprising a hollow shaft 851 and a drain tube 853.

The body 891 includes a first end 891 a, a second end 891 b, and aconduit 892 running through the body 891 from the first end 891 a to thesecond end 891 b. At the first end 891 a, the conduit 892 is coupled(e.g., by press-fit, and adhesive, or by a threaded connection) to thedrain tube 853 of the plunger. At the second end 891 b, the conduit 892opens into a recessed opening 894. Thus, the recessed opening 894 isfluidically connected to the conduit 892 and the drain tube 853.

The body 891 may be fabricated from plastic (e.g., polypropylene,polyethylene, polytetrafluoroethylene) by molding, for example. The body891 is shaped and dimensioned to fit in a housing (e.g., housing 710 ofFIG. 7A). In any embodiment, the body 891 or the O-ring 886 can form asubstantially liquid-tight seal with the walls of a housing when thebody 891 is inserted into the housing. In any embodiment that includesan O-ring, 886, the O-ring 886 may function both to form a liquid-tightseal and to wipe particulate material (e.g., cell concentration agents)off the wall of the housing as the O-ring 886 is moved in relation tothe wall of the housing. The shaft 851 may be coupled to the conduit 892by means that are known in the art (e.g., by an adhesive, by press-fit).The optional O-ring 886 is disposed in a notch 889 in the body 891.

The tip 890 further comprises a filter 896. The filter 896 is coupled tothe body 891 at the recessed opening 894. In the illustrated embodiment,the filter 896 is formed from a porous material, which can be press-fitand/or adhesively coupled to the recessed opening 894. In someembodiments, the porous material can be semi-rigid porous material(e.g., POREX filtration medium sold under the part number X6854 by PorexCorporation, Fairburn, Ga.). The filter 896 may be configured with arelatively angular or pointed end, such that the end can facilitate thepenetration of a frangible seal. In alternative embodiments (not shown),the filter may comprise a membrane filter that is coupled to the body.When coupled to the body, the membrane filter is part of a fluid paththat includes the conduit and a drain channel.

In some embodiments, the porosity of the filter 896 may be selected suchthat the filter 896 prevents only the passage of relatively largeparticles (e.g., >1 μm, >5 μm, or >10 μm,) through it. Relatively largeparticles may include, for example, cell concentration agents asdescribed herein. In these embodiments, microorganisms such as bacteria,yeast, and/or filamentous fungi (mold) may pass through the filter 896.

In some embodiments, the porosity of the filter 896 may be selected suchthat the filter 896 prevents only the passage of relatively smallparticles (e.g., <1 μm, <0.45 μm, <0.2 μm,) through it. In theseembodiments, microorganisms such as bacteria, yeast, and/or filamentousfungi (mold) may be retained by the filter 896.

The tip 890 may further comprise an optional one-way valve 897 disposedin the recessed opening 894 between the filter 896 and the conduit 892.Also shown is an optional retaining washer 898 that can serve to holdthe one-way valve 897 in position. The one-way valve 897 may beconstructed from plastic (e.g., polypropylene, polyethylene, polyester)or rubber, for example, and may be configured as a duck-bill valve, forexample. In use, the one-way valve 897 substantially prevents the flowof liquid that has passed through the filter 896 from returning throughthe filter 896 in the opposite direction.

FIG. 8B shows a side view, partially in section of the assembled tip 790of FIG. 7A. The one-way valve 897, optional retaining washer 898, andfilter 896 are disposed in the recessed opening and are in fluidicconnection with the conduit 892 and the drain tube 853. The shaft 851 iscoupled to the body 891 of the tip 890.

Referring back to FIG. 7A, the detection device 700 comprising thehousing 710 and plunger 750 is used in a method to detect microorganismsand, in particular, live microorganisms.

In use, a liquid sample is transferred into the upper receptacle 720 ofthe housing 710, where it is allowed to contact a cell concentrationagent 730. After adding the liquid sample 740 to the housing 710, thetip 790 of the plunger 750 is inserted into the housing 710 and urged(e.g., manually or mechanically) toward the lower receptacle 724 of thehousing 710, as shown in FIG. 7B. As the tip 791 of the plunger 750contacts the liquid sample 740, the liquid passes through the tip 790,through the drain tube 753, and into the hollow shaft of the plunger750, as shown in FIG. 7B. This process retains the cell concentrationagent 730 and, in some embodiments, free microorganisms in a portion 742of the liquid sample proximate the third receptacle 726.

As the tip 790 of the plunger 750 penetrates the frangible seal 760 a,not shown, the portion 742 of the liquid sample containing the cellconcentration agent 730 contacts the hydrogel 762. Further movement ofthe plunger 750 (as shown in FIG. 7D) causes penetration of thefrangible seal 760 b, which causes the portion 742 of the liquid sampleand the hydrogel 762 to transfer to the lower receptacle 724, where theycontact the detection reagent 765.

Devices of the present disclosure include frangible seals in a housing.The frangible seals are pierced to transport the cell concentrationagent from one compartment of the device to another compartment. In someembodiments, the amount cell concentration agent transferred in thatprocess can be enhanced by collecting the cell concentration agent ontoa relatively area of the frangible seal. FIG. 9 shows one embodiment ofa collector 967 to enhance the recovery of cell concentration agent. Thecollector 967 is dimensioned to fit within the housing of a detectiondevice according to the present disclosure. The collector 967 comprisesa beveled edge 968 that is oriented toward the sample comprising a cellconcentration agent (not shown). Typically, the beveled edge 968 facesupward such that it collects particles that are settling by the force ofgravity. Alternatively, the beveled edge 968 could be oriented toward acentrifugal or a hydrodynamic force, for example, to collect particlessubjected to forces other than gravity. The collector 967 furthercomprises an optional frangible seal 969.

The collector 967 can be fabricated from a variety of materialsincluding, for example a polymer (e.g., polyester, polypropylene,polytetrafluoroethylene, polypropylene, polystyrene, nylon, andcombinations and derivatives thereof), glass, and metal. The collector967 may further comprise a lubricious coating to resist the adherence ofparticles to its surface. The beveled edge 968 may be angled (e.g. a45-degree angle, a >45-degree angle) to facilitate the movement ofparticles down its slope. The frangible seal 969 is fabricated asdescribed herein and may be coupled to the collector 967 by means thatare described herein.

FIG. 10A shows one embodiment of a detection device 1000 comprising acollector 1067. The device 1000 comprises a housing 1010 and a plunger1050. The housing 1010 comprises an upper part 1012 and a lower part1014. Disposed within the upper part 1012 and proximate the lower part1014 is a collector 1067 with a frangible seal 1069 coupled thereto. Thefrangible seal 1069 is coupled to the side of the collector 1067 that isfacing the lower part 1014. Thus, the frangible seal 1069 divides thehousing 1010 into two isolated receptacles, an upper receptacle 1020 anda lower receptacle 1024.

The plunger 1050 comprises a handle 1052, a shaft 1051, and a tip 1090.The handle 1052 can be constructed as described above and may comprisean optional rim 1054 that engages the housing 1010 to prevent theplunger 1050 from being inserted too far into the housing 1010. Thehandle 1052 can be coupled to the shaft 1051 via a threaded fit or byother coupling means (e.g., press-fit, adhesive). The tip 1090 may befabricated fusing processes and materials described for other tipembodiments described herein. The tip 1090 may comprise one or moreguides 1083. The guides are dimensioned to loosely fit within theinterior of the housing 1010 and function to reduce lateral movement ofthe tip 1090 as the tip 1090 moves longitudinally through the housing1010.

The tip 1090 further comprises a scraper 1086, which is held in a fixedposition on the tip 1090. In the illustrated embodiment, the scraper1086 is held in a fixed position by retaining member 1087. The retainingmember 1087 can be molded or machined as part of the tip 1090 or it cancomprise a bracket or plurality of brackets coupled to the tip 1090.Alternatively, the scraper 1086 may be directly coupled (e.g.,adhesively coupled) to the tip 1090.

The scraper 1086 is disc-shaped and is dimensioned to form a relativelytight fit inside the housing 1010. In some embodiments, the scraper cancomprise an-O-ring. The scraper 1086 should substantially maintain itsshape when immersed in an aqueous liquid. Although the scraper should bedimensioned to form a relatively tight fit inside the housing, thescraper should be relatively flexible to permit fluid to flow around itsedge as the plunger is pushed through a liquid sample in the housing1010. Suitable materials for fabricating the scraper 1086 include, forexample polyurethane rubber.

In use, a liquid sample 1040 and a cell concentration agent 1030 arecontacted in the housing 1010 of the device, as shown in FIG. 10B. Theplunger 1050 is inserted into the housing 1010 and the tip 1090 of theplunger 1050 is urged toward the bottom of the housing 1010. As the tip1090 passes through the liquid sample 1040, the cell concentration agent1030 is urged toward the bottom of the housing 1010 by the scraper 1086,while the liquid sample 1040 flows around the edge of the scraper 1086.Advantageously, this devices allows the user to collect and concentratethe cell concentration agent 1030 in a substantially shorter period oftime than possible if the cell concentration agent 1030 is allowed tosettle by gravity force to the bottom of the housing 1010. Furthermore,the flexible scraper facilitates collecting a portion of cellconcentration agent 1030 that might otherwise adhere to the walls of thehousing. Thus, the inventive device 1000 increases the recovery of thecell concentration agent and, thereby, increases the sensitivity of amethod that uses a cell concentration agent to concentratemicroorganisms.

It should be recognized that, in a sample preparation and detectiondevice in which the cell concentration agent comprises ferromagneticmaterials (e.g., particles), that a magnet or an electromagnet can bepositioned adjacent the device to draw the particles (and microorganismscoupled thereto) to a desired location for collecting the particlesand/or transferring them to another receptacle. In some embodiments, themagnet can be positioned adjacent the device (e.g., adjacent the bottomof the device) after a sufficient period of time to allow for the cellconcentration agent to couple substantially all of the microorganisms inthe liquid sample.

Methods of Detecting Biological Analytes from Live Cells:

Methods of the present disclosure include methods for the detection ofbiological analytes that are released from live cells including, forexample, live microorganisms, after exposure to an effective amount ofcell extractant.

Methods of the present disclosure include the formation of a liquidmixture comprising a sample suspected of containing live cells and ahydrogel comprising a cell extractant. Methods of the present disclosurefurther include detecting a biological analyte. Detecting a biologicalanalyte can further comprise quantitating the amount of biologicalanalyte in the sample.

In one aspect, the present disclosure provides a method of detectingcells in a sample. The method comprises providing a cell concentrationagent, a hydrogel comprising a cell extractant and a liquid samplesuspected of containing cells. Suitable cell concentration agents aredescribed in U.S. Patent Application No. 60/977,180 filed on Oct. 3,2007, and entitled “MICROORGANISM CONCENTRATION PROCESS”, which isincorporated herein by reference in its entirety.

The method further comprises contacting the liquid sample and the cellconcentration agent for a period of time. The cell concentration agentcan comprise particles, fibers, a matrix (e.g., a fibrous matrix)comprising particles, or any combination of two or more of theforegoing. The cell concentration agent can be suspended in the liquidsample during the contact period. The suspension can be placed into avessel, such as a tube, a flask, a beaker, or any of the detectiondevices described herein. In certain preferred embodiments, the liquidsample is mixed with the cell concentration agent for a period of timeby, for example, stirring, vortexing, or vibrating the suspension. Whilethe cell concentration agent is contacted with the liquid sample, cellsfrom the liquid sample are coupled to the cell concentration agent.

The method further comprises isolating the cell concentration agent fromat least a portion of the liquid sample. During this process, the cellconcentration agent may be concentrated in a smaller volume than theoriginal liquid sample. The cell concentration agent can be isolatedfrom at least a portion of the liquid sample by a variety of means. Forexample, if the cell concentration agent has a higher specific gravitythan the liquid sample, the cell concentration agent can settle to thebottom of the suspension. At least a portion of the liquid sample can beremoved (e.g., by pipetting or decanting). Alternatively, oradditionally, at least a portion of the liquid sample can be removed bycentrifugation or filtration.

A filter can be described by its pore size (for example by its bubblepoint pore size). The bubble point pore size of a filter is generallythe average of the largest size of the pores of the filter. In someembodiments, the filter can have an average pore size that is less thanthe average size of the cell concentration agent. The ability to utilizefilters having these relatively large pore sizes offers significantadvantages to methods as disclosed herein when compared with othermethods for separating microorganisms from samples, such as watersamples.

In an embodiment, the filter can have an average pore size that is atleast about 1 micrometer (μm) or larger. In an embodiment, the filtercan have an average pore size that is at least about 1.5 μm or larger.In an embodiment, the filter can have an average pore size that is atleast about 5 μm or larger. In an embodiment, the filter can have anaverage pore size that is at least about 10 μm or larger. As larger poresize filters are utilized, the sample will be easier and quicker tofilter as the back pressure decreases with increase in pore size.

Filtering the sample can be accomplished using known methods. In anembodiment, the method of filtering that is chosen can be dictated atleast in part on the particular application of the method. For example,the sample can be filtered using a negative vacuum, by applying apositive pressure, by the force of gravity. The particular techniqueused to filter the sample can depend at least in part on the type ofdevice that is being utilized to carry out the method. For example, inorder to utilize a negative vacuum, the device can be configured with aport that can be or reversibly attached to a source of vacuum; and inorder to apply a positive pressure, the device can be configured toallow a user to apply a positive pressure by applying a force with theirhands. In an embodiment, the sample can be filtered by applying apositive pressure.

Filtering using positive pressure (or using the force of gravity) canoffer the advantage of easily being able to carry out the method in thefield without the need for any further equipment, such as a vacuum pump.

In some embodiments, a centrifugation step may include the use of arelatively low-speed centrifugation in which the cell concentrationagents separate (e.g., by sedimentation) out of the liquid butmicroorganisms (e.g., bacteria, yeast molds, spore) that are not boundto the cell concentration agent remain suspended in the liquid.

Optionally, the cell concentration agent can be resuspended in a washsolution (e.g., water or a buffer solution) and the cell concentrationagent can be isolated from at least a portion of the wash solution. Itwill be recognized that a washing step can function to remove from theliquid sample contaminating materials that may interfere with a growthand/or detection process.

The method further comprises forming a liquid mixture comprising theisolated cell concentration agent and the hydrogel, wherein the cellextractant is released from the hydrogel. In some embodiments, when thecell concentration agent is isolated from at least a portion of theliquid sample, the cell concentration agent remains in a residual volumeof liquid. Additional liquid (e.g., water or a buffer solution)optionally can be added to the cell concentration agent. In theembodiments wherein the cell concentration agent is filtered out of theliquid sample, the cell concentration agent can be resuspended in avolume of liquid (e.g., water or a buffer solution). The liquidsuspension comprising the cell concentration agent is contacted with thehydrogel, thereby releasing the cell extractant into the liquid mixture.In methods involving the use of filters to collect the cellconcentration agent, the liquid suspension comprising the cellconcentration agent can also comprise the filter. An effective amount ofcell extractant can be released from the hydrogel to effect the releaseof biological analytes from cells, if present, in the mixture. Therelease of an effective amount of cell extractant can occur over aperiod of time (e.g., up to several seconds, up to several minutes, upto an hour, or longer).

The method further comprises detecting an analyte. The detection of thebiological analytes can involve the use of a detection system. Detectionsystems for certain biological analytes such as a nucleotide (e.g., ATP,NADH, NAD), a polynucleotide (e.g., DNA or RNA) or an enzyme (e.g., NADHdehydrogenase or adenylate kinase) are known in the art and can be usedaccording to the present disclosure. Methods of the present disclosureinclude known detections systems for detecting a biological analyte.Preferably, the accuracy and sensitivity of the detection system is notsignificantly reduced by the cell extractant. More preferably, thedetection system comprises a homogeneous assay.

In some embodiments, detecting the biological analyte can comprisedetecting the analyte directly in a vessel (e.g., a tube, a multi-wellplate, and the detection devices described herein) in which the liquidmixture comprising the sample and the hydrogel comprising a cellextractant is formed. In some embodiments, detecting the biologicalanalyte can comprise transferring at least a portion of the liquidmixture to a container other than the vessel in which the liquid mixturecomprising the sample and the hydrogel comprising a cell extractant isformed. In some embodiments, detecting the biological analyte maycomprise one or more sample preparation processes, such as pHadjustment, dilution, filtration, centrifugation, extraction, and thelike.

In some embodiments, the detection system comprises a detection reagent.Detection reagents include, for example, dyes, enzymes, enzymesubstrates, binding partners (e.g., an antibody, a monoclonal antibody,a lectin, a receptor), labeled binding partners, and/or cofactors. Insome embodiments, the detection reagent comprises a hydrogel, such asthe hydrogels comprising an enzyme or enzyme substrate, as described inU.S. Patent Application No. 61/101,546, filed on Sep. 30, 2008, andentitled “BIODETECTION ARTICLES”. In some embodiments, the detectionsystem comprises an instrument. Nonlimiting examples of detectioninstruments include a spectrophotometer, a luminometer, a plate reader,a thermocycler, an incubator.

Detection systems can include detection instruments. Detectioninstruments are known in the art and can be used to detect biologicalanalytes colorimetrically (i.e., by the absorbance and/or scattering oflight), fluorescently, or lumimetrically. Examples of the detection ofbiomolecules by luminescence are described by F. Gorus and E. Schram(Applications of bio- and chemiluminescence in the clinical laboratory,1979, Clin. Chem. 25:512-519).

An example of a biological analyte detection system is an ATP detectionsystem. The ATP detection system can comprise an enzyme (e.g.,luciferase) and an enzyme substrate (e.g., luciferin). The ATP detectionsystem can further comprise a luminometer. In some embodiments, theluminometer can comprise a bench top luminometer such as, for example,the FB-12 single tube luminometer (Berthold Detection Systems USA, OakRidge, Tenn.). In some embodiments, the luminometer can comprise ahandheld luminometer such as, for example, the NG Luminometer, UNG2 (3MCompany, St. Paul, Minn.).

In some embodiments, the biological analyte is detected at a single timepoint. In some embodiments, the biological analyte is detected at two ormore time points. When the biological analyte is detected at two or moretime points, the amount of biological analyte detected at a first time(e.g., before an effective amount of cell extractant is released from ahydrogel to effect the release of biological analytes from live cells inat least a portion of the sample) point can be compared to the amount ofbiological analyte detected at a second time point (e.g., after aneffective amount of cell extractant is released from a hydrogel toeffect the release of biological analytes from live cells in at least aportion of the sample). In some embodiments, the measurement of thebiological analyte at one or more time points is performed by aninstrument with a processor. In certain preferred embodiments, comparingthe amount of biological analyte at a first time point with the amountof biological analyte at a second time point is performed by theprocessor.

For example, the operator measures the amount of biological analyte inthe sample after the liquid mixture including the sample and thehydrogel comprising a cell extractant is formed. The amount ofbiological analyte in this first measurement (T₁) can indicate thepresence of “free” (i.e. acellular) biological analyte and/or biologicalanalyte from nonviable cells in the sample. In some embodiments, thefirst measurement can be made immediately (e.g., about 1 second) afterthe liquid mixture including the sample and the hydrogel comprising acell extractant is formed. In some embodiments, the first measurementcan be at least about 5 seconds, at least about 10 seconds, at leastabout 20 seconds, at least about 30 seconds, at least about 40 seconds,at least about 60 seconds, at least about 80 seconds, at least about 100seconds, at least about 120 seconds, at least about 150 seconds, atleast about 180 seconds, at least about 240 seconds, at least about 5minutes, at least about 10 minutes, at least about 20 minutes after theliquid mixture including the sample and the hydrogel comprising a cellextractant is formed. These times are exemplary and include only thetime up to that the detection of a biological analyte is initiated.Initiating the detection of a biological analyte may include dilutingthe sample and/or adding a reagent to inhibit the activity of the cellextractant. It will be recognized that certain detection systems (e.g.,nucleic acid amplification or ELISA) can generally take several minutesto several hours to complete.

The operator allows the sample to contact the hydrogel comprising thecell extractant for a period of time after the first measurement ofbiological analyte has been made. After the sample has contacted thehydrogel for a period of time, a second measurement of the biologicalanalyte is made. In some embodiments, the second measurement can be madeup to about 0.5 seconds, up to about 1 second, up to about 5 seconds, upto about 10 seconds, up to about 20 seconds, up to about 30 seconds, upto about 40 seconds, up to about 60 seconds, up to about 90 seconds, upto about 120 seconds, up to about 180 seconds, about 300 seconds, atleast about 10 minutes, at least about 20 minutes, at least about 60minutes or longer after the first measurement of the biological analyte.These times are exemplary and include only the interval of time fromwhich the first measurement for detecting the biological analyte isinitiated and the time at which the second measurement for detecting thebiological analyte is initiated. Initiating the detection of abiological analyte may include diluting the sample and/or adding areagent to inhibit the activity of the cell extractant.

Preferably, the first measurement of a biological analyte is made about1 seconds to about 240 seconds after the liquid mixture including thesample and the hydrogel comprising a cell extractant is formed and thesecond measurement, which is made after the first measurement, is madeabout 1.5 seconds to about 540 seconds after the liquid mixture isformed. More preferably, the first measurement of a biological analyteis made about 1 second to about 180 seconds after the liquid mixture isformed and the second measurement, which is made after the firstmeasurement, is made about 1.5 seconds to about 120 seconds after theliquid mixture is formed. Most preferably, the first measurement of abiological analyte is made about 1 second to about 5 seconds after theliquid mixture is formed and the second measurement, which is made afterthe first measurement, is made about 1.5 seconds to about 10 secondsafter the liquid mixture is formed.

The operator compares the amount of a biological analyte detected in thefirst measurement to the amount of biological analyte detected in thesecond measurement. An increase in the amount of biological analytedetected in the second measurement is indicative of the presence of oneor more live cells in the sample.

In certain methods, it may be desirable to detect the presence of livesomatic cells (e.g., nonmicrobial cells). In these embodiments, thehydrogel comprises a cell extractant that selectively releasesbiological analytes from somatic cells. Nonlimiting examples of somaticcell extractants include nonionic detergents, such as non-ionicethoxylated alkylphenols, including but not limited to the ethoxylatedoctylphenol Triton X-100 (TX-100) and other ethoxylated alkylphenols;betaine detergents, such as carboxypropylbetaine (CB-18), NP-40, TWEEN,Tergitol, Igepal, commercially available M-NRS (Celsis, Chicago, Ill.),M-PER (Pierce, Rockford, Ill.), CelLytic M (Sigma Aldrich). Cellextractants are preferably chosen not to inactivate the analyte and itsdetection reagents.

In certain methods, it may be desirable to detect the presence of livemicrobial cells. In these embodiments, the hydrogel can comprise a cellextractant that selectively releases biological analytes from microbialcells. Nonlimiting examples of microbial cell extractants includequaternary ammonium compounds, including benzalkonium chloride,benzethonium chloride, ‘cetrimide’ (a mixture of dodecyl-, tetradecyl-and hexadecyl-trimethylammonium bromide), cetylpyridium chloride;amines, such as triethylamine (TEA) and triethanolamine (TeolA);bis-Biguanides, including chlorhexidine, alexidine and polyhexamethylenebiguanide dialkyl ammonium salts, includingN-(n-dodecyl)-diethanolamine, antibiotics, such as polymyxin B (e.g.,polymyxin B1 and polymyxin B2), polymyxin-beta-nonapeptide (PMBN);alkylglucoside or alkylthioglucoside, such asOctyl-β-D-1-thioglucopyranoside (see U.S. Pat. No. 6,174,704 hereinincorporated by reference in its entirety); nonionic detergents, such asnon-ionic ethoxylated alkylphenols, including but not limited to theethoxylated octylphenol Triton X-100 (TX-100) and other ethoxylatedalkylphenols; betaine detergents, such as carboxypropylbetaine (CB-18);and cationic, antibacterial, pore forming, membrane-active, and/or cellwall-active polymers, such as polylysine, nisin, magainin, melittin,phospholipase A₂, phospholipase A₂ activating peptide (PLAP);bacteriophage; and the like. See e.g., Morbe et al., Microbiol. Res.(1997) vol. 152, pp. 385-394, and U.S. Pat. No. 4,303,752 disclosingionic surface active compounds which are incorporated herein byreference in their entirety. Cell extractants are preferably chosen notto inactivate the biological analyte and/or a detection reagent used todetect the biological analyte.

In certain alternative methods to detect the presence of live microbialcells in a sample, the sample can be pretreated with a somatic cellextractant for a period of time (e.g., the sample is contacted with asomatic cell extractant for a sufficient period of time to extractsomatic cells before a liquid mixture including the sample and ahydrogel comprising a microbial cell extractant is formed). In thealternative embodiment, the amount of biological analyte detected at thefirst measurement will include any biological analyte that was releasedby the somatic cells and the amount of additional biological analyte, ifany, detected in the second measurement will include biological analytefrom live microbial cells in the sample.

Methods to detect the presence of a microorganism in a sample caninclude the use of the detection devices disclosed herein. In certainembodiments, the method comprises providing i) a sample suspected ofcontaining cells, ii) a detection article comprising a housing with twoor more receptacles and an opening configured to receive the sample,iii) a cell concentration agent, and iv) a means for isolating andtransferring the cell concentration agent from a upper receptacle to alower receptacle in the housing, and v) a hydrogel comprising a cellextractant. In these embodiments, the detection device can comprise anyone of the detection devices 100, 200, 300, or 400, shown in FIGS. 1-4.Optionally, the detection device can comprise the cell concentrationagent and/or the hydrogel.

The method further comprises transferring the sample into an upperreceptacle in the housing wherein, in a liquid medium, the samplematerial is contacted with the cell concentration agent. The sample cancomprise liquids, solids, semi-solids, or combinations thereof, whichare transferred into the upper receptacle of the housing. If the sampledoes not comprise a liquid medium, a liquid medium (e.g., water or abuffered solution) can be added to the upper receptacle. A cellconcentration agent is added to the liquid sample. The cellconcentration agent is allowed to contact the liquid sample for a periodof time. Optionally, the mixture can be mixed during the contact periodby, for example, shaking, stirring, vortexing, and/or vibrating thehousing. Preferably, the housing is closed (e.g., with optional cap)during the contact period to avoid loss of the sample and/or cellconcentration agent.

The method further comprises isolating, from at least a portion of theliquid medium, the cell concentration agent, wherein isolating the cellconcentration agent comprises transferring the cell concentration agentto a lower receptacle in the housing. As described herein, there are avariety of means for isolating the cell concentration agent.Non-limiting examples of means to isolate and transfer the cellconcentration agent include partitioning and transferring the cellconcentration agent through a passageway using a plunger (see FIGS. 1and 2), collecting and transferring the cell concentration agent in thecavity of a one-way valve (see FIG. 3), and concentrating andtransferring the cell concentration agent using a drain valve and aplunger (see FIG. 4).

The method further comprises forming a liquid mixture comprising theisolated cell concentration agent and the hydrogel, wherein the cellextractant is released into the mixture. The liquid mixture comprisingthe cell concentration agent is contacted with a hydrogel comprising acell extractant. The hydrogel (e.g., a hydrogel bead) can be contactedwith the liquid mixture in the upper receptacle and/or lower receptacleof the housing. In some embodiments, the lower receptacle of the housingcontains the hydrogel (see FIGS. 1 and 3) and the liquid mixture iscontacted with the hydrogel when the mixture is transferred into thelower receptacle. In some embodiments, the hydrogel is disposed in athird receptacle (see FIGS. 2 and 4), through which the liquid samplepasses (thereby contacting the liquid sample with the hydrogel) as theliquid sample is transferred from the upper receptacle to the lowerreceptacle.

It is recognized that, although FIGS. 2 and 4 show the use of a plungerto pierce the frangible seals and transfer the cell concentration agentto the lower receptacle, alternative instruments (e.g., a swab, apipette, a filter) could be used instead of a plunger. In a method wheresuch alternative instruments are used, it is preferable to remove atleast a portion of the liquid sample (e.g., by decanting, pipetting,filtering, or by opening the drain valve, if present) such that theentire liquid sample is not transferred to the second receptacle whenthe frangible seal is pierced by the alternative instrument.

The method further comprises detecting a biological analyte. Thebiological analyte can be detected, as described herein, in the lowerreceptacle of the detection device before an effective amount of cellextractant is released from the hydrogel into the liquid mixturecomprising the cell concentration agent. The biological analyte can bedetected, as described herein, in the lower receptacle of the detectiondevice after an effective amount of cell extractant is released from thehydrogel into the liquid mixture comprising the cell concentrationagent. The biological analyte can be detected, as described herein, inthe lower receptacle of the detection device before and after aneffective amount of cell extractant is released from the hydrogel intothe liquid mixture comprising the cell concentration agent.

It is anticipated that any of the methods disclosed herein can furthercomprise a biological growth step. The growth step is facilitated byproviding a nutrient medium to support the growth of a microorganism.The nutrient medium can be mixed with the sample before, during, orafter the concentration of microorganisms by the cell concentrationagent. In some embodiments, the biological growth step occurs after themicroorganisms have been concentrated by the cell concentration agentbut before the biological analyte is detected. In some embodiments, thenutrient medium can contain nutrients and/or selective agents (e.g.,salts, antibiotics) that favor the growth of certain types ofmicroorganisms over other microorganisms that may be present in thesample.

Method of Concentrating a Particulate Cell Concentration Agent:

The present disclosure provides devices for concentrating a particulatecell concentration agent. The method includes providing a device toseparate a portion of a liquid sample from a suspension the particulatematerial in the liquid sample. Suitable devices include, for example,the devices shown and described in FIG. 2A, FIG. 3A, FIG. 4A, FIG. 5A,FIG. 7A, and FIG. 10A. The devices each comprise a housing to contain aliquid sample including a particulate cell concentration agent and ameans for separating the particulate cell concentration agent from atleast a portion of the liquid sample.

In FIG. 2A, the means for separating the particulate cell concentrationagent includes the taper region 218 and the plunger comprising a lowerseal 256. In FIG. 3A, the means for separating the particulate cellconcentration agent includes the dead-end valve 370 and valve actuator372. In FIG. 4A, the means for separating the particulate cellconcentration agent includes the drain valve 480 and valve gate 482. InFIGS. 5A and 7A, the means for separating the particulate cellconcentration agent includes the plunger comprising fluid path with afilter 596 disposed in the fluid path. In FIG. 10A, the means forseparating the particulate cell concentration agent includes the plungerwith a scraper that is configured to permit the passage of liquidbetween the edge of the scraper and the housing.

The method further comprises forming a suspension of particulate cellconcentration agent in a liquid sample. The suspension may be formed inthe housing or it may be formed outside the housing. If the suspensionis formed outside of the housing, the method further comprisestransferring the suspension into the housing. The method furthercomprises contacting the particulate cell concentration agent with theliquid sample for a period of time sufficient to capture amicroorganism. The contacting may occur in the housing. The contactingmay occur outside the housing. The contacting may occur both outside andinside the housing. The method further comprises separating a portion ofa liquid sample from a suspension the particulate material in the liquidsample, as described above for the devices of FIGS. 2A, 3A, 4A, 5A, 7A,and 10A.

Sample Preparation and Detection Kits:

Components and/or devices of the present disclosure can be packagedtogether with instructions and optionally, accessory articles orreagents, to produce sample preparation and detection kits. Thus, in oneaspect, the present disclosure provides a kit comprising i) a housingcomprising at least two receptacles with a passageway therebetween, ii)means for isolating a upper receptacle from a lower receptacle in thehousing, iii) a cell concentration agent, and iv) means for transferringthe cell concentration agent from the upper receptacle to the lowerreceptacle. The upper receptacle comprises an opening configured toreceive a sample. The lower receptacle comprises a detection reagentdisposed therein. In some embodiments, the housing can further comprisethe means for isolating the upper receptacle from the lower receptacle,as described herein. In some embodiments, the housing can furthercomprise the means for transferring the cell concentration agent fromthe upper receptacle to the lower receptacle. In some embodiments, thecell concentration agent is disposed in the upper receptacle of thehousing.

In some embodiments, kits of the present disclosure include accessoryarticles or reagents that can be used with the sample preparation anddetection devices. Nonlimiting examples of accessory articles include asample acquisition device, a filter, a glove, a culture device (e.g., apetri plate, a culture tube, a PETRIFILM plate obtained from 3M Company(St. Paul, Minn.), or the like), nucleic acid isolation or amplificationreagents, immunoassay devices such as lateral flow devices, ELISA platesand reagents, or any combination of two or more of the foregoingarticles. Nonlimiting examples of accessory reagents include water, abuffering agent, an indicator (e.g., a pH indicator), a dye, a somaticcell extractant, a hydrogel comprising a cell extractant, a bindingpartner as described herein, an enzyme, an enzyme substrate,oligonucleotides, control samples or any combination of two or more ofthe foregoing reagents.

EXAMPLES

The present invention has now been described with reference to severalspecific embodiments foreseen by the inventor for which enablingdescriptions are available. Insubstantial modifications of theinvention, including modifications not presently foreseen, maynonetheless constitute equivalents thereto. Thus, the scope of thepresent invention should not be limited by the details and structuresdescribed herein, but rather solely by the following claims, andequivalents thereto.

Materials:

All bacterial cultures were obtained from The American Type CultureCollection (ATCC, Manassas, Va.), unless specified otherwise.

All water was obtained as 18 megaohm sterile deionized water using aMilli-Q™ Gradient deionization system from Millipore Corporation(Bedford, Mass.), unless specified otherwise.

CM-111: amorphous, spheroidized magnesium silicate; microspheres wereobtained as 3M™ Cosmetic Microspheres CM-111 from 3M Company, St. Paul,Minn. The particles were shaped as solid spheres with particle densityof 2.3 g/cc and had a surface area approximately 3.3 m²/g. Ninetypercent of the particles were less than about 11 microns. Fifty percentof the particles were less than about 5 microns. Ten percent of theparticles were less than about 2 microns. CM-111 microspheres wereprepared as described in Example 1 of U.S. Patent Application No.61/289,213, filed on Dec. 22, 2009 and entitled “MICROORGANISMCONCENTRATION PROCESS AND CONCENTRATION AGENT FOR USE THEREIN”, which isincorporated herein by reference in its entirety.

The 100× adsorption buffer containing 500 mM KCl, 100 mM CaCl₂, 10 mMMgCl₂, and 100 mM K₂HPO₄ at pH 7.2 was prepared and filter-sterilizedprior to use.

Surface-sterilized components were contacted (wiped with or immersed in)70% isopropyl alcohol. The excess alcohol was poured off and thecomponents were allowed to air-dry for at least 30 minutes before use.

All chemicals were obtained from Sigma-Aldrich Chemical Company,Milwaukee, Wis., unless specified otherwise.

Example 1 Incorporation of Cell Extractant into Hydrogel Beads afterPolymerization of the Hydrogel

Hydrogel beads were prepared as described in example 1 of theInternational Patent Publication No. WO 2007/146722. Active beads wereprepared by drying as described in example 19 and then soaking in activesolution as described in example 23 of the International PatentPublication No. WO 2007/146722. One gram of beads was dried at 60° C.for 2 h to remove water from the beads. The dried beads were soaked in 2grams of 50% (w/v) aqueous solutions of BARDAC 205M (Lonza Group Ltd.,Valais, Switzerland) for at least 3 hrs to overnight at roomtemperature. After soaking, the beads were poured into a Buchner funnelto drain the beads and then rinsed with 10 to 20 ml of distilled water.The excess water was removed from the surface of the beads by blottingthem with a paper towel. The beads were stored in a jar at roomtemperature for at least two weeks before they were used.

Example 2 Cell Concentration by Use of Microparticles and DetectionUsing Cell Extractant-Loaded Hydrogels and ATP Bioluminescence

3M™ CLEAN-TRACE Surface ATP system was obtained from 3M Company (St.Paul, Minn.). Pure cultures of E. coli ATCC 51183 were inoculated intotryptic soy broth and grown overnight at 37° C. The bacterial culturewas diluted to approximately 10⁶ or 10⁵CFU/ml in Butterfield's buffer(pH 7.2±0.2; monobasic potassium phosphate buffer solution; VWR, WestChester, Pa.) and 100 microliters of the diluted suspension were addeddirectly to individual tubes containing ten ml of deionized water(Milli-Q Biocel System, Millipore, MA) samples to obtain approximately10⁵ CFU or 10⁴ CFU in ten ml, respectively. Ten mg of autoclaved CM-1113M™ Cosmetic Microspheres (calcined amorphous spheroidized magnesiumsilicate powders; 3M Company; St. Paul, Minn.) were added to the tubescontaining cells and mixed at room temperature for about 15 min. Theparticles were allowed to settle and the supernatant was removed. Theparticles were suspended in 100 μl of Butterfield's buffer andtransferred to 1.5 ml microfuge tubes. Four hundred microliters ofluciferase/luciferin liquid reagent solution from CLEAN-TRACE surfaceATP system was added to the tubes. For the control (unconcentrated)reactions, 100 μl of approximately 10⁶ or 10⁵ CFU/ml cell suspensionwere added to 1.5 ml microfuge tubes and 400 μl of luciferase/luciferinliquid reagent solution from Clean-Trace surface ATP system was added tothe tubes. Immediately after adding the reagent, hydrogel beads (about11 mg) containing a cell extractant were added to individual tubes andrelative light units (RLUs) measurements were recorded at 10 secintervals in a bench-top luminometer (20/20n single tube luminometerfrom Turner Biosystems, Sunnyvale, Calif.). Luminescence measurementswere obtained from the luminometer using 20/20n SIS software that wasprovided with the luminometer. The light signal was integrated for 1second and the results, expressed in RLU, are presented in Table 1.

The data indicate that, using the protocol described herein, themicroparticles were able to concentrate the microbial cells and the cellextractant released from the hydrogel beads was able to extract ATP fromE. coli. The results further indicate that ATP released from the cellsreacted with the ATP-detection reagents, which resulted in measurablebioluminescence.

TABLE 1 Detection of ATP from E. coli cells coupled to a cellconcentration agent and exposed to microbial cell extractants releasedfrom a hydrogel. Values expressed in the table are relative light units(RLUs). Time Unconcentrated Concentrated (sec) 10⁴ Cfu 10⁵ Cfu 10⁴ Cfu10⁵ Cfu 10 1226 2552 904 1648 20 1239 2648 948 1735 30 1265 2681 10001735 40 1272 2820 1067 1786 50 1280 3152 1107 1818 60 1312 3914 11471948 70 1352 4960 1178 2139 80 1393 6391 1197 2388 90 1440 8258 12262732 100 1538 10230 1250 3188 120 1618 11859 1260 3820 130 1704 129691286 4681 140 1838 13527 1297 5842 150 1905 13759 1318 6721 160 200613735 1309 6675 170 2088 13762 1314 6513 180 2119 13537 1330 6428 1902169 13426 1363 6321 200 2140 13353 1375 6220 210 2141 13128 1342 6196220 2143 13014 1389 6142 230 2155 12903 1381 6076 240 2110 12780 14016023

Example 3 Detection of Microbial Cells in a Unitary Sample Preparationand Detection Device Using an ATP Bioluminescence Detection System

A unitary sample preparation and detection device 200, as shown in FIG.2, is used in this Example. The device contains approximately 10 mg ofautoclaved CM-111 3M Cosmetic Microspheres in the upper receptacle 220.Lower receptacle 224 contains a liquid detection reagent 265, whichconsists of approximately 0.6 milliliters of the luciferase/luciferinliquid reagent solution from a CLEAN-TRACE surface ATP system. The thirdreceptacle 226 contains two BARDAC 205M beads made according toPreparative Example 5 of U.S. Patent Application No. 61/101,546, filedSep. 30, 2008. Ten milliliters of sterile deionized water is added tothe upper receptacle 220 of the unitary devices 200 immediately beforeuse.

E. coli overnight cultures are prepared as described in Example 2. Thebacterial culture is diluted to approximately 10⁶ or 10⁵ CFU/ml inButterfield's buffer. One hundred microliters of the diluted suspensionare pipetted directly into upper receptacle 220 of the unitary devices200 to obtain a suspension of approximately 10⁵ CFU or 10⁴ CFU in tenmilliliters, respectively. The cap 278 is used to close the housing 210and the bacterial suspension is mixed with the microspheres (cellconcentration agent 230) at room temperature and allowed to settle intothe passageway 216. The cap 278 is removed and the plunger 250 isinserted to transfer a portion of the liquid sample containing thesettled microspheres and hydrogel beads into the lower receptacle 224,which contains the ATP detection reagents. The unitary device isimmediately inserted into the reading chamber of a luminometer (forexample, a NG Luminometer, UNG2) and RLU measurements are recorded at 10sec interval using the Unplanned Testing mode of the UNG2 luminometer.RLU measurements are collected until the number of RLUs reaches aplateau. The data are downloaded using the software provided with the NGluminometer. The data will indicate that the microbial cells areconcentrated by the microspheres, the cell extractant is released by thehydrogel, the cell extractant causes the release of ATP from the cells,and the ATP released from the cells is detected by the ATP detectionsystem.

Example 4 Detection of Microbial Cells in a Unitary Sample Preparationand Detection Device Using an ATP Bioluminescence Detection System

A unitary sample preparation and detection device 300, as shown in FIG.3, is used in this Example. The valve actuator 372 is positioned suchthat the valve cavity 374 is in fluid communication with the upperreceptacle 320 prior to use. The device contains approximately 10 mg ofautoclaved CM-111 3M Cosmetic Microspheres in the upper receptacle 320.Lower receptacle 324 contains a liquid detection reagent 365, whichconsists of approximately 0.6 milliliters of the luciferase/luciferinliquid reagent solution from a Clean-Trace surface ATP system. BARDAC205M beads are made according to Preparative Example 5 of U.S. PatentApplication No. 61/101,546, filed Sep. 30, 2008. Ten milliliters ofsterile deionized water is added to the upper receptacle 320 of theunitary devices 300 immediately before use.

E. coli overnight cultures are prepared as described in Example 2. Thebacterial culture is diluted to approximately 10⁶ or 10⁵ CFU/ml inButterfield's buffer. One hundred microliters of the diluted suspensionare pipetted directly into upper receptacle 320 of the unitary devices300 to obtain a suspension of approximately 10⁵ CFU or 10⁴ CFU in tenmilliliters, respectively. The cap 378 is used to close the housing 310and the bacterial suspension is mixed with the microspheres (cellconcentration agent 330) at room temperature and allowed to settle intothe valve cavity 374. The cap 378 is removed and two BARDAC 205M beads(hydrogel 362) are dropped into the housing 310. Immediately after thebeads settle into the valve cavity 374, the valve actuator 372 is turnedto transfer the portion of the liquid sample in the valve cavity(containing the cell concentration agent 330 and the hydrogel 362) intothe lower receptacle 324 containing the ATP detection reagents. Theunitary device is immediately inserted into the reading chamber of aluminometer (for example, a NG Luminometer, UNG2) and RLU measurementsare recorded at 10 sec interval using the Unplanned Testing mode of theUNG2 luminometer. RLU measurements are collected until the number ofRLUs reaches a plateau. The data are downloaded using the softwareprovided with the NG luminometer. The data will indicate that themicrobial cells are concentrated by the microspheres, the cellextractant is released by the hydrogel, the cell extractant causes therelease of ATP from the cells, and the ATP released from the cells isdetected by the ATP detection system.

Example 5 Preparation of Detection Devices

Type I devices: For these detection devices, housings similar to thehousing of FIG. 10A were constructed with the differences noted below.Reference numbers below refer to the corresponding parts in FIG. 10A.The upper parts 1012 and lower parts 1014 of the housing 1100 wereobtained using the analogous components from 3M Clean-Trace™ surface ATPtests (obtained from 3M Company, Bridgend, UK). A collector 1067 with afrangible seal 1068 coupled thereto was press-fit into the upper portionof the lower part 1014; with the frangible seal 1068 facing the lowerpart 1014 of the housing 1100. The upper part 1012 was coupled to thelower part 1014 using a 2 cm section of 3:1 polyolefin dual walladhesive lined heat shrink film obtained from buyheatshrink.com (part#_HSC3A-050-cc, 1.5 cm in diameter) using a heat gun (Master AppliancesCorp, Racine, Wis.).

For these detection devices, plungers similar to the plunger of FIG. 2Awere constructed. Reference numbers below refer to the correspondingparts in FIG. 2A. The plunger (250) was assembled using a portion of thepolyolefin plastic handle (252) from a 3M Clean-Trace™ surface ATP test,a brass metal shaft (251) and an acetal piercing member 259. The handle252 and piercing member 259 were attached to the ends of the brass shaftvia threaded connections. The brass metal shaft was 11.5 cm long and 3.9mm in diameter. A 6 mm, 6-23 thread was produced on each end of theshaft using a lathe. The piercing member 259 was fabricated from ½-inch(12.7 mm) acetal copolymer rod (part number 8497K211, obtained fromMcMASTER-CARR, Santa Fe Springs, Calif.) using a 10″ Southbend lathe. AnO ring (Buna N AS568A Dash Number 010 obtained from McMASTER-CARR) wasused as the lower seal 256 and was attached to the plunger 250approximately 11.5 mm above the piercing end 259. The plunger wassurface-sterilized before each use.

Type II devices: These detection devices were assembled using a plungersimilar to that shown and described in FIG. 5A with a tip similar tothat shown in FIG. 6A. The housing was constructed as described for theType I devices. The tip of the plunger was fabricated from ½-inch (12.7mm) acetal copolymer rod (part number 8497K211, obtained fromMcMASTER-CARR, Santa Fe Springs, Calif.) using a 10″ Southbend lathe.The a duckbill one-way valve and a plastic retaining washer werepress-fit into the recessed opening of the body of the tip of theplunger. The filter was made by machining a POREX filter (part numberX6854 from Porex Corporation, Fairburn, Ga.) to the shape shown in FIG.6A and dimensioning one end to press-fit into the recessed opening ofthe tip and hold the valve and retaining washer in place. The plungerwas surface-sterilized before each use.

Type III devices: Detection devices similar to those shown in FIG. 10Awere constructed with the differences noted below. Reference numbersbelow refer to the corresponding parts in FIG. 10A. The upper parts 1012and lower parts 1014 of the housing 1100 were obtained using theanalogous components from 3M Clean-Trace™ surface ATP tests (obtainedfrom 3M Company, Bridgend, UK). A collector 1067 with a frangible seal1068 coupled thereto was press-fit into the upper portion of the lowerpart 514; with the frangible seal 1068 facing the lower part 1014 of thehousing 1100. The upper part 1012 was coupled to the lower part 1014using a 2 cm section of 3:1 polyolefin dual wall adhesive lined heatshrink film obtained from buyheatshrink.com (part #_HSC3A-050-cc, 1.5 cmin diameter) using a heat gun (Master Appliances Corp, Racine, Wis.).

The plunger (1050) was assembled using a portion of the polyolefinplastic handle (1052) from a 3M Clean-Trace™ surface ATP test, a brassmetal shaft (1051) and tip 1090. The handle 1052 and tip 1090 wereattached to the ends of the brass shaft via threaded connections. Thebrass metal shaft was 11.5 cm long and 3.9 mm in diameter. A 6 mm, 6-23thread was produced on each end of the shaft using a lathe. The tip 1090was fabricated from ½-inch (12.7 mm) acetal copolymer rod (part number8497K211, obtained from McMASTER-CARR, Santa Fe Springs, Calif.) using a10″Southbend lathe. An O ring 1086 was attached to the tip 1090. The tipwas machined to include a retaining member 1087, as shown in FIG. 10. Ascraper was constructed by die-cutting a piece of 1 mm-thickpolyurethane rubber and slipping it into the retaining member 1087. Theouter diameter of the scraper 1086 was dimensioned to provide a tightfit with the inside of the housing 1010. The plunger wassurface-sterilized before each use.

Example 6 Capture of E. coli from Spiked Water with ParticulateConcentration Agents Using a Type I Device

An isolated colony of E. coli (ATCC 51813) from a Tryptic Soy Agar plate(Becton Dickinson, Sparks, Md.) was used to inoculate 5 ml Tryptic SoyBroth (Becton Dickinson, Sparks, Md.) and incubated overnight in a 37°C. incubator. The overnight culture containing approximately 10⁹ colonyforming units/ml (CFU/ml) was diluted 1:10,000 (to approximately 10⁵CFU/mL, hereinafter called “initial diluted suspension”) in filtersterilized 18 megaohm water. Five hundred microliters of the dilutedculture were transferred to 50 ml of filter sterilized 18 megaohm water,resulting in a final concentration of about approximately 1000/ml.

An aliquot (0.5 mL) of 100× Adsorption Buffer (pH 7.2) was added to the50 mL diluted E. coli suspension (hereinafter called “spiked watersample”). The contents were mixed by manual mixing for about a minute.

An amount of 10 mg of steam sterilized CM-111 was weighed and added toType I devices prepared as described in example 5. A 10 ml volume of thespiked water sample was added to each device and the devices were cappedwith surface sterilized Para film. The contents were mixed by shakingmanually at room temperature (25° C.) for about 30 seconds.

After mixing, the devices were incubated for various time periods (1, 5,10 and 20 minutes, respectively) on a Thermolyne Vari Mix™ rockingplatform (Barnstead International, Iowa, 14 cycles/minute). After theincubation the tubes were set on the bench top for 10 minutes to settlethe particulate concentration agent, CM-111. After settling, Para filmwrapping was removed and the pre-sterilized plunger device was used topierce the foil seal and deposit the settled CM-111 agent into the lowerpart of the device. The supernatant was removed by using a pipette, andthe lower part of the device (which contained the cell concentrationagent) was separated from the upper part of the device using a razorblade. The settled CM-111 concentration agent (in approximately 100microliters of water) was removed from the device; diluted 1:100 insterile water, and one-milliliter aliquots of the diluted concentrationagent were plated on 3M™ Petrifilm™ Aerobic Count Plate (3M Company, St.Paul, Minn.) according to the manufacturer's instructions.

As a control, the initial diluted suspension was further diluted 1:1000dilution in sterile water and was plated as on 3M™ Petrifilm™ AerobicCount Plate (3M Company, St. Paul, Minn.) according to themanufacturer's instructions. The particulate materials were also platedon Petrifilm™ Aerobic Count Plate as sterility controls. The plates wereincubated overnight in a 37° C. incubator (VWR Orbital Shaker Incubator,VWR, West Chester, Pa.).

All plates were analyzed by using 3M™ Petrifilm™ Plate Reader (3MCompany, St. Paul, Minn.) according to the manufacturer's instructionsand colony counts were obtained. The results are shown in Table 2. Theresults were calculated using the following formula:

Capture efficiency=(Number of colonies on concentration agent/TotalNumber of colonies in the spiked control)×100

TABLE 2 Concentration/capture of E. coli from 10 ml sample. All datarepresent the average of two replicate tests per experiment. % SampleControl Stdev  1 min 8 4  5 min 34 4 10 min 33 11 20 min 80 10

Example 7 Concentration of E. coli Using CM-111 Using a Type III Device

An isolated E. coli (ATCC 51813) colony was inoculated from a streakplate into 5 ml Tryptic Soy Broth (TSB, Becton Dickinson, Sparks, Md.)and incubated at 37° C. for 18-20 hours. This overnight culture at ˜10 ⁹colony forming units/ml was diluted in sterile-filtered deionized water(MilliQ, Millipore, MA) and spiked in 10 ml of sterile-filtereddeionized water to obtain final concentration of 1×10³/ml and 1×10⁴/ml(˜1×10⁴/ml cfus and ˜1×10⁵/ml cfus total). The spiked water was added tothe housing of a Type III device containing 10 mg pre-sterilized (121deg C., 15 minutes) powder of CM-111 (Cosmetic Microspheres-111, 3MCompany, St Paul) and 100 microliters of the 100× Adsorption Buffer (pH7.2). The housing was sealed with surface sterilized Parafilm and placedon a rocking platform The capped devices were then incubated at roomtemperature (25° C.) for 5 minutes contact time on a Thermolyne VariMix™ rocking platform (Barnstead International, Iowa, 14 cycles/minute).The devices were then allowed to stand without shaking (to allow theparticles to settle by gravity force) for 5 minutes (total elapsed timefor rocking and settling=10 minutes), the Parafilm was removed and theType II device plunger inserted into the housing and was urged towardthe bottom of the housing to separate the CM-111 particles from the bulksample. When the plunger broke the frangible seal, the CM-111 particles,suspended in about 0.1 mL of the liquid sample, was transferred to thelower receptacle of the housing. The CM-111 particles were retrievedfrom the lower receptacle and transferred to a 1.5 ml sterile microfugetube. A 100 microliter volume of the BacTiter-Glo™ reagent (Promega,Madison, Wis.) was added to the pellet, mixed by vortexing for 5 secondson a VWR Fixed Speed Vortex Mixer (3200 rpm for 5 seconds) and read on atabletop luminometer (FB12 Single Tube Luminometer, Berthold DetectionSystems USA, Oak Ridge, Tenn.). A positive control (“100% signal”) wasprepared by testing a 100 microliter volume from a 1×10⁵/ml and 1×10⁶/mlsuspension of the E. coli cells. Results were calculated using theformula below and tabulated in Table 2 below:

ATP Signal % Capture efficiency=(RLUs on CM-111 pellet/RLUS from 100%signal)×100

RLU=Relative Luciferase Units.

TABLE 2 ATP signal ATP signal Capture Sample in RLUs efficiency (%) E.coli (1 × 10⁴ cells) control 30,866 N/A (100% signal) E. coli (1 × 10⁵cells) control 176,933 N/A (100% signal) CM-111 pellet from ~1 × 10³/ml27,589 89 sample CM-111 pellet from ~1 × 10⁴/ml 94,840 54 sample N = 2,Std deviation <10%, Data normalized to water alone (16,464 RLUs)background for E. coli controls and normalized to unreacted CM-111(41,424 RLUs) background for the CM-111 pellets contacted with bacteria.

From above example it can be seen that the particulate capture agentscan be used to concentrate bacteria from an aqueous sample.

Example 8 Concentration of E. coli Using CM-111 Using a Type II Device

An isolated E. coli (ATCC 33090) colony was inoculated from a streakplate into 5 ml Tryptic Soy Broth (, TSB, Becton Dickinson, Sparks, Md.)and incubated at 37° C. for 18-20 hours. This overnight culture atapproximately 10⁸ colony forming units/ml was diluted insterile-filtered deionized water (MilliQ, Millipore, MA) and spiked in10 ml of sterile-filtered deionized water to obtain final concentrationof 10³/ml (approximately 10⁴ cfus total). The spiked water was added tothe device already containing 10 mg pre-sterilized (121 deg C., 15minutes) powder of CM-111 (Cosmetic Microspheres-111, 3M Company, StPaul) and 100 microliters of the 100× Adsorption Buffer. The device wassealed with surface sterilized Parafilm and placed on a rocking platformThe capped devices were then incubated at room temperature (25° C.) for1 and 9 minutes (total elapsed=time 2 mins and 10 minutes) on aThermolyne Vari Mix™ rocking platform (Barnstead International, Iowa, 14cycles/minute).

After the incubation the Parafilm was removed and the plunger with thewas inserted into the housing until it contacted the frangible seal. Byinserting the plunger further, to break the frangible seal, the E. colibound CM-111 was transferred to the lower receptacle of the housingalong with approximately 100 microliters of the liquid sample. Controltubes containing E. coli without microparticles were treated similarly.

The CM-111 pellet was retrieved by cutting open the lower receptacle theparticles were transferred to a 1.5 ml sterile microfuge tube. A 100microliter volume of the BacTiter-Glo™ reagent (Promega, Madison, Wis.)was added to the pellet, mixed by vortexing for 5 seconds on a VWR FixedSpeed Vortex Mixer (3200 rpm for 5 seconds) and read on a tabletopluminometer (FB12 Single Tube Luminometer, Berthold Detection SystemsUSA, Oak Ridge, Tenn.). For 100% signal, a 100 microliter volume from a10⁵/ml dilution was used. Results were calculated using the formulabelow and tabulated in Table 3 below:

ATP Signal % Capture efficiency ═(RLUs on CM-111 pellet/RLUS from about10⁴ total E. coli)×100

RLU=Relative Luciferase Units.

TABLE 3 ATP signal ATP signal Capture Sample in RLUs efficiency (%) E.coli (10⁴ cells) control 96,544 N/A Water sample with E. coli (no 25,583 0 concentration) CM-111 pellet with concentrated 56,932 59 E. coli 2min testing time CM-111 pellet with concentrated 58,543 61 E. coli 10min testing time N = 2, Std deviation <10%, Data normalized to wateralone (27,938 RLUs) background for E. coli controls and normalized tounreacted CM-111 (30,611 RLUs) background for the CM-111 pelletscontacted with bacteria.

Example 9 Concentration of E. coli Using AB-CM-111 Using a Type IIDevice

A 10 mg aliquot of AB-CM (Adsorption buffer treated CM-111) was alsotested for concentration of E. coli from 10 ml water using the proceduredescribed in Example 8. The contact time was 9 minutes, 1 min to settleAB-CM using the POREX plunger. The data is tabulated in Table 4.

TABLE 4 ATP signal in ATP signal Capture Sample RLUs efficiency (%) E.coli (10⁴) control 82,845 N/A Water sample with E. coli (no 733  1concentration) AB-CM pellet with concentrated 44,105 53 E. coli N = 2,Std deviation <10%, Data normalized to water alone (20,281 RLUs)background for E. coli controls and normalized to unreacted AB-CM(44,488 RLUs) background for the CM-111 pellets contacted with bacteria.

From above example it can be seen that the particulate capture agentscan be used to concentrate bacteria from an aqueous sample.

Example 10 Comparative Example Detection of E. coli in UnconcentratedSamples

State-of-the-art water testing comprises a method where 100 microlitersof water is tested for ATP using a standard ATP bioluminescence assay(for example, 3M CLEANTRACE Water—Free ATP Cat. No. AQF100, availablefrom 3M Company, St. Paul, Minn.).

An overnight culture of E. coli (ATCC 33090) in tryptic soy broth wasdiluted in sterile water to produce two suspensions. Suspension Acontained approximately 10³ CFU/ml and Suspension B contained about 10⁵CFU/ml. One hundred microliter aliquots of each suspension were mixedwith 100 microliter volumes of the BacTiter-Glo™ reagent (Promega,Madison, Wis.) and the resulting bioluminescence was measured with aluminometer as described in Example 8. The results are presented inTable 5.

TABLE 5 ATP signal Capture ATP signal in efficiency (%) after SampleRLUs normalizing to water E. coli (10⁴) control (100% 18,143 N/A Signal)Water sample (no E. coli) 1,109 N/A Water sample with E. coli (no 1,7764% concentration) N = 2, Std deviation <10%

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A method of detecting cells in a sample, the method comprising:providing a cell concentration agent, a hydrogel comprising a cellextractant and a liquid sample suspected of containing cells; contactingthe liquid sample and the cell concentration agent for a period of time;isolating the cell concentration agent from at least a portion of theliquid sample; forming a liquid mixture comprising the isolated cellconcentration agent and the hydrogel, wherein the cell extractant isreleased into the mixture; and detecting a biological analyte; whereindetecting the biological analyte comprises quantifying an amount of thebiological analyte; wherein the amount of the biological analyte isquantified two or more times; wherein the amount of biological analytedetected at a first time point is compared to the amount of biologicalanalyte detected at a second time point.
 2. A method of detecting cellsin a sample, the method comprising: providing a sample suspected ofcontaining cells; a cell concentration agent; a hydrogel comprising acell extractant; a detection article comprising a housing with two ormore receptacles and an opening configured to receive the sample; andmeans for isolating and transferring the cell concentration agent froman upper receptacle to a lower receptacle in the housing; contacting ina liquid medium the sample with the cell concentration agent in theupper receptacle of the housing; isolating and transferring the cellconcentration agent to the lower receptacle in the housing; forming aliquid mixture comprising the isolated cell concentration agent and thehydrogel, wherein the cell extractant is released into the mixture; anddetecting a biological analyte; wherein detecting the biological analytecomprises quantifying an amount of the biological analyte; wherein theamount of the biological analyte is quantified two or more times;wherein the amount of biological analyte detected at a first time pointis compared to the amount of biological analyte detected at a secondtime point.
 3. A method of detecting cells in a sample, the methodcomprising: providing a sample suspected of containing cells; adetection article comprising a housing with an opening configured toreceive the sample, an upper receptacle containing a cell concentrationagent, and a lower receptacle containing a hydrogel comprising a cellextractant; means for isolating the cell concentration agent from atleast a portion of the liquid sample; and means for transferring thecell concentration agent from the upper receptacle to the lowerreceptacle in the housing; contacting in a liquid medium the sample andthe cell concentration agent in the upper receptacle of the housing;isolating and transferring the cell concentration agent to the lowerreceptacle of the housing; forming a liquid mixture comprising theisolated cell concentration agent and the hydrogel, wherein the cellextractant is released into the mixture; and detecting a biologicalanalyte; wherein detecting the biological analyte comprises quantifyingan amount of the biological analyte; wherein the amount of thebiological analyte is quantified two or more times; wherein the amountof biological analyte detected at a first time point is compared to theamount of biological analyte detected at a second time point. 4-10.(canceled)
 11. The method of claim, wherein detecting the biologicalanalyte comprises detecting ATP from cells.
 12. The method of claim 11,wherein detecting the ATP from cells comprises detecting ATP frommicrobial cells. 13-15. (canceled)
 16. The method of claim 1, whereindetecting the biological analyte comprises detecting the biologicalanalyte immunologically.
 17. The method of claim 1, wherein detectingthe biological analyte comprises detecting the biological analytegenetically.
 18. The method of claim 1, wherein detecting the biologicalanalyte comprises detecting an enzyme released from a live cell in thesample.
 19. The method of claim 18, wherein the enzyme comprisesadenylate kinase enzyme activity.
 20. (canceled)
 21. A unitary samplepreparation and detection device, comprising: a housing comprising atleast two receptacles with a passageway therebetween; wherein an upperreceptacle comprises an opening configured to receive a sample and acell concentration agent disposed therein; wherein a lower receptacleincludes a detection reagent disposed therein; means for isolating theupper receptacle from the lower receptacle; and means for transferringthe cell concentration agent from the upper receptacle to the lowerreceptacle.
 22. The device of claim 21, wherein the cell concentrationagent comprises a particulate or dispersed cell concentration agent. 23.The device of claim 21, wherein the means for isolating the upper andlower receptacles of the housing includes a plunger, a valve, or afrangible seal.
 24. The device of claim 21, wherein the means fortransferring the cell concentration agent from the upper receptacle tothe lower receptacle of the housing includes a plunger, a swab, or avalve.
 25. The device of claim 21, wherein the device comprises atapered inner wall.
 26. The device of claim 21, further comprising ahydrogel comprising a cell extractant. 27-28. (canceled)
 29. The deviceof claim 21, wherein the housing further comprises a third receptacle.30-31. (canceled)
 32. The device of claim 21, further comprising aplunger.
 33. The device of claim 32, wherein the plunger comprises afluid pathway. 34-37. (canceled)
 38. A kit comprising: a housingcomprising at least two receptacles with a passageway therebetween;wherein an upper receptacle comprises an opening configured to receive asample; wherein a lower receptacle comprises a detection reagentdisposed therein; means for isolating the upper receptacle from thelower receptacle; a cell concentration agent; and means for transferringthe cell concentration agent from the upper receptacle to the lowerreceptacle.
 39. The kit of claim 38, wherein the housing comprises themeans for isolating the upper receptacle from the lower receptacle. 40.The kit of claim 38, wherein the housing comprises the means fortransferring the cell concentration agent from the upper receptacle tothe lower receptacle.
 41. The kit of claim 38, wherein the cellconcentration agent is disposed in the upper receptacle of the housing.42. (canceled)
 43. The kit of claim 38, further comprising a hydrogelcomprising a microbial cell extractant. 44-45. (canceled)
 46. The kit ofclaim 38, further comprising a plunger.
 47. The kit of claim 46, whereinthe plunger comprises a fluid pathway. 48-51. (canceled)